DWF4 polynucleotides, polypeptides and uses thereof

ABSTRACT

The present invention relates to novel, polynucleotides isolated from dwarf plants. The dwf4 polynucleotides that encode all, or a portion of, a DWF4 polypeptide, a cytochrome P450 enzyme that mediates multiple steps in synthesis of brassinosteroids. The present invention also relates to isolated polynucleotides that encode regulatory regions of dwf4. Uses of the dwf4 polypeptides and polynucleotides are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to provisional patent applicationsSer. No. 60/119,657, filed Feb. 11, 1999 and 60/119,658, filed Feb. 11,1999, from which priority is claimed under 35 USC §119(e)(1) and whichapplications are incorporated herein by reference in their entireties.

TECHNICAL FIELD

[0002] The present invention relates to novel polynucleotides isolatedfrom dwarf plants. The dwf4 polynucleotides encode all, or a portion of,a DWF4 polypeptide, a cytochrome P450 enzyme that mediates multiplesteps in synthesis of brassinosteroids. The present invention alsorelates to isolated polynucleotides that encode regulatory regions ofdwf4. Uses of the dwf4 polypeptides and polynucleotides are alsodisclosed.

BACKGROUND

[0003] Plant growth is accomplished by orderly cell division and tightlyregulated cell expansion. In plants, the contribution of cell expansionto growth is of much greater significance than in most other organisms;all plant organs owe their final size to a period of significant cellelongation, which usually follows active cell division. Further, thesessile nature of plants requires that they make fine but responsiveadjustments in growth to survive harsh environmental conditions and tooptimize their use of limited resources (Trewavas (1986) “Resourceallocation under poor growth conditions: A major role for growthsubstances in developmental plasticity” In Plasticity in Plants, D. H.Jennings and A. J. Trewavas, eds (Cambridge, UK: Company of BiologistsLtd.), pp. 31-76).

[0004] In Arabidopsis, cell elongation is largely responsible forhypocotyl growth in germinating seedlings and extension ofinflorescences (bolting) at the end of vegetative growth. Coordinatecontrol of plant growth is regulated by both external stimuli andinternal mechanisms. Of the external signals, the most obvious is light(Deng, X.-W. (1994) Cell 76:423-426). Light inhibits hypocotylelongation and promotes cotyledon expansion and leaf development inseedlings, and photoperiod is crucial for flower initiation in a largenumber of species.

[0005] The internal components of plant signaling are generally mediatedby chemical growth regulators (phytohormones; reviewed in Klee, H., andEstelle, M. (1991) Annu. Rev. Plant Physiol. Plant Mol. Biol.42:529-551). Thus, plant growth in response to environmental factors ismodulated by plant hormones acting alone or in concert (Evans “Functionsof hormones at the cellular level of organization” In HormonalRegulation of Plant Physiology, T. K. Scott, ed (Berlin:Springer-Verlag), pp. 23-79), and growth depends on regulated cellularevents, such as division, elongation, and differentiation.

[0006] Gibberellic acid (GA) and cytokinins promote flowering; inaddition, GA stimulates stem elongation, whereas cytokinins have theopposite effect, reducing apical dominance by stimulating increasedaxillary shoot formation. Conversely, auxins promote apical dominanceand stimulate elongation by a process postulated to requireacidification of the cell wall by a K⁻-dependent H⁺-pumping ATPase(Rayle, D. L., and Cleland, R. E. (1977) Curr. Top. Dev. Biol.11:187-214).

[0007] In addition to the classic hormones, such as auxin andgibberellic acid (GA), brassinosteroids (BRs) have been discovered to beimportant in growth promotion (reviewed in Clouse (1996) Plant J.10:1-8). The most recently discovered class of plant growth substances,the BRs, has been to date the least studied; however, rapid progresstoward understanding BR biosynthesis and regulation is now being made(Yokota, T. (1997) Trends Plant Sci. 2:137-143). The term BRscollectively refers to the growth-promoting steroids found in plants(Grove et al. (1979) Nature 281:216-217). They are structurally verysimilar to the molting hormones of insects, ecdysteroids (Richter andKoolman (1991) “Antiecdysteroid effects of brassinosteroids in insects”in Brassinosteroids: Chemistry, Bioactivity, and Applications, H. G.Cutler, T. Yokota, and G. Adam, eds (Washington, D.C.: American ChemicalSociety), pp. 265-279), but active BRs have unique structural features.As shown in FIG. 1, a 6-oxolactone or 7-oxalactone in the B ring, 5αhydrogen, and multiple hydroxylations at four different positions withspecific stereochemistry have been proposed as an essentialconfiguration for BRs (reviewed in Marquardt and Adam (1991) “Recentadvances in brassinosteroid research” in Chemistry of Plant Protection,W. Ebing, ed (Berlin: Springer-Verlag), pp. 103-139). Among >40naturally occurring BRs, brassinolide (BL; 2α, 3α, 22(R),23(R)-tetrahydroxy-24(S)-methyl-B-homo-7-oxa-5α-cholestan-6-one) hasbeen shown to be the most biologically active (reviewed in Mandava(1988) Annu. Rev. Plant Physiol. Plant Mol. Biol. 39:23-52). As a majorbiological effect, BRs stimulate longitudinal growth of young tissuesvia cell elongation and cell division (reviewed in Clouse (1996), supra;Fujioka and Sakurai (1997a) Nat. Prod. Rep. 14:1-10).

[0008] Elucidating the BR biosynthetic pathways has been a major area ofrecent interest. Biochemical analyses have been used to elucidate the BRbiosynthetic pathway (Fujioka et al. (1996) Plant Cell Physiol.37:1201-1203; Choi et al. (19-97), Phytochemistry 44:609-613), andmutational analyses are being used to confirm this pathway. Similar tothe biosynthetic pathways of the human steroid hormones and insectecdysteroids (Rees (1985) “Biosynthesis of ecdysone” in ComprehensiveInsect Physiology, Biochemistry and Pharmacology, G. A. Kerkut and L. I.Gilbert, eds (Oxford, UK: Pergamon Press), pp. 249-293; Granner, D. K.(1996) “Hormones of the gonads” in Harper's Biochemistry, R. K. Murray,D. K. Granner, P. A. Mayes, and V. W. Rodwell, eds (Stamford, Conn.:Appleton and Lange Press), pp. 566-580), BRs are synthesized viamultiple parallel pathways (Fujioka et al. (1996) Plant Cell Physiol.37:1201-1203; Choi et al. (1997), supra). Starting from the initialprecursor, campesterol (CR), the BR intermediates undergo a series ofhydroxylations, reductions, an epimerization, and a Baeyer-Villigerûtypeoxidation leading to the most oxidized form, BL (Fujioka and Sakurai(1997b) Physiol. Plant. 100:710-715; FIG. 1). Castasterone (CS)oxidation, the last step in BR biosynthesis, is not found in somespecies, such as mung bean. In that case, CS plays a role as the majorBR rather than BL (Yokota et al. (1991) “Metabolism and biosynthesis ofbrassinosteroids” in Brassinosteroids: Chemistry, Bioactivity, andApplication, H. G. Cutler, T. Yokota, and G. Adam, eds (Washington,D.C.: American Chemical Society), pp. 86-96). Traditionally, BRbiosynthetic pathways have been elucidated by feeding deuterio-labeledintermediates to BR-producing cell lines of Madagascar periwinkle(Sakurai and Fujioka (1996) “Catharanthus roseus (Vinca rosea): In vitroproduction of brassinosteroids” in Biotechnology in Agriculture andForestry, Y. P. S. Bajaj, ed (Berlin: Springer-Verlag), pp. 87-96.). Thepresent model, including parallel branched pathways and early and lateC-6 oxidation pathways, was established using these feeding studies(Fujioka and Sakurai (1997a), supra, Fujioka and Sakurai (1997b), supra;Sakurai and Fujioka (1997) Biosci. Biotechnol. Biochem. 61:757-762).

[0009] Although the brassinosteriod system is a less well understoodclass of plant growth substances (BRs; Mitchell, et al. (1970) Nature225:1065-1066; Grove et al. (1979) Nature 281:216-217; Mandava, N. B.(1988) Annu. Rev. Plant Physiol. Plant Mol. Biol. 39:23-52), severalsuch compounds have been identified and are known to effect elongationof cells in various plant tissues, their biosynthesis, regulation, andmechanism of action have only recently begun to be elucidated (reviewedin Clouse, S. D. (1996) Plant J. 10:1-8; Fujioka, S., and Sakurai, A.(1997) Physiol. Plant. 100:710-715).

[0010] Several types of dwarf or dwarflike mutants have been describedin Arabidopsis. A number of mutations have been identified that affecteither light-dependent (cop, det, and fusca [fus; another group ofmutants with some members perturbed in light-regulated growth]) orhormone signaling (axr2) pathways and whose pleiotropic phenotypesinclude defects in cell elongation. The majority of these mutants alsohave other alterations in their phenotypes. At least five GA mutantshave been described as being reduced in stature (Koornneef and Van derVeen (1980) Theor. Appl. Genet. 58:257-263). GA biosynthetic mutants mayalso have no or defective flower development and are marked by anabsence of viable pollen. Reduced levels of endogenous gibberellins arealso a characteristic (Barendse et al.(1986) Physiol. Plant. 67:315-319;Talon et al. (1990) Proc. Natl. Acad. Sci. USA 87:7983-7987), and theirphenotype can be nearly restored to that of the wild type by theaddition of exogenous GA. (Koornneef and Van der Veen (1980) Theor.Appl. Genet. 58:257-263).

[0011] Another hormone mutation, auxin resistant2 (axr2), results inplants with a dwarf phenotype both in the light and in darkness as wellas increased resistance to high levels of auxin, ethylene, and abscisicacid (Timpte et al. (1992) Planta 188:271-278). An interestingrelationship exists between light regulation and cytokinin levels.Arabidopsis seedlings grown in the dark in the presence of cytokininshave open cotyledons, initiate chloroplast differentiation and leafdevelopment, and activate transcription from the chlorophyll a/b bindingprotein gene (CAB) promoter. Importantly, they also display a cytokinindose-dependent dwarf phenotype.

[0012] Dwarf Arabidopsis mutants that are rescued by addition of BRshave also been described (Kauschmann et al. (1996) Plant J 9:701-713; Liet al. (1996) Science 272:398-401; Szekeres et al. (1996) Cell85:171-182; Azpiroz et al. (1998) Plant Cell 10:219-230), including thefollowing three mutants: dwarfl (dwfl; Kauschmann et al. (1996) Plant J.9:701-713), constitutive photomorphogenesis and dwarfism (cpd; Szekereset al. (1996) Cell 85:171-182), and det2 (Li et al. (1996) Science272:398-401). These mutants have been shown to be defective in steroidbiosynthesis. DWF1 (Feldmann et al. (1989) Science 243:1351-1354) wascloned first (GenBank accession number U12400). Takahashi et al. (1995)Genes Dev. 9:97-107 hypothesized that DWF1, which they isolated with anallele of dwf1, referred to as diminuto1 (dim1), contains a potentialnuclear targeting signal, which may confer a regulatory function to theprotein. However, Mushegian and Koonin (1995) Protein Sci. 4:1243-1244indicated that DWF1 displays limited homology with flavin adeninedinucleotide (FAD)independent oxidoreductase, suggesting an enzymaticfunction in BR biosynthesis. According to Kauschmann et al. (1996),supra (dwf1-6 described as cabbage1 [cbb1]), dwf1 mutants were rescuedby exogenous application of BRs.

[0013] DET2 was shown to encode a putative steroid 5α-reductase,mediating an early step in BR biosynthesis (Li et al. (1996), supra, Liet al. (1997) Proc. Natl. Acad. Sci. USA 94:3554-3559; Fujioka et al.(1997) Plant Cell 9:1951-1962; FIG. 1). Moreover, det1 and det2 have adecreased requirement for cytokinins in tissue culture and appear to besaturated for a cytokinin-dependent delay in senescence (Chory et al.(1994) Plant Physiol. 104:339-347). CPD has been proposed to be a novelcytochrome P450 (CYP90A1; Szekeres et al. (1996), supra), encoding aputative 23α-hydroxylase that acts in BR biosynthesis. The range ofphenotypes in the deetiolated (det) and constitutive photomorphogenic(cop) light-regulatory mutants is broad. Mutations in DET1, COP1, COP8,COP9, COP10, and COP11 result in constitutive derepression ofsubstantial portions of the photomorphogenic program (Chory, et al.(1989b) Cell 58:991-999; Deng, X.-W., and Quail, P. H. (1992) Plant J.2:83-95; Wei, N., and Deng, X.-W. (1992) Plant Cell 4:1507-1518; Wei etal. (1994) Plant Cell 6:629-643), whereas mutations in COP4 seem toaffect only morphology and gene expression (Hou et al. (1993) Plant Cell5:329-339). The only invariant phenotype in this class oflight-regulatory mutants is a substantial reduction in height in bothlight and darkness.

[0014] There are additional dwarfs that are insensitive to one of thesehormones, such as bri (brassinosteroid insensitive; Clouse et al. (1996)Plant Physiol. 111:671-678; Li and Chory (1997) Cell 90:929-938), gai(gibberellic acid insensitive; Koornneef et al. (1985) Physiol. Plant.65:33-39), and axr2 (auxin resistant2; Timpte et al. (1994) Genetics138:1239-1249). Clouse et al. (1996), supra isolated bri by screeningethyl methanesulfonate-mutagenized populations for mutants whose rootgrowth is not retarded at inhibitory concentrations of BR. Thus, the BRIprotein is proposed to be involved in BR signal perception ortransduction (Clouse (1996), supra). Kauschmann et al. (1996), supradescribed a phenotypically similar mutant cbb2 that maps to the samelocation. In addition, the dwf2 alleles possess a phenotype similar tobri and map to the same region (Feldmann and Azpiroz (1994) “dwarf(dwj)and twisted dwarf (twd)” in Arabidopsis: An Atlas of Morphology andDevelopment, J. Bowman, ed (New York: Springer-Verlag), pp. 82-85). Itseems likely that all of the BR-insensitive dwarf mutants described todate are allelic. Recently, BRI has been cloned and shown to encode aleucine-rich-repeat receptor kinase, suggesting a role in the BR signaltransduction pathway (Li and Chory (1997), supra).

[0015] Mutants defective in BR biosynthesis have also been isolated inother plant species. Bishop et al. (1996) Plant Cell 8:959-969 isolateda tomato dwarf mutant by transposon tagging. The tomato Dwarf geneencodes a pioneering member of the CYP85 family, and it appears to beinvolved in BR biosynthesis. In addition, Nomura et al. (1997) PlantPhysiol. 113:31-37 reported that the lka and lkb mutants in garden peaare deficient in BR biosynthesis (lkb) or perception (lka).

[0016] Currently, little is known about the downstream events that occurin response to these signals and thereby directly control cell size.This is because the biochemical and cell biological processes involvedhave thus far been difficult to address. In addition, there is littleinformation about the integration of regulatory signals converging atthe cell from different signaling pathways and the ways they arecoordinately controlled. In particular, the interaction of light andhormones in the control of cell elongation is not clear. Thus, thereremains a need for the identification and characterization of additionalmutants and polypeptides encoded thereby involved in these pathways ofplant growth.

SUMMARY OF THE INVENTION

[0017] In one aspect the invention includes an isolated dwf4polynucleotide comprising an open reading frame that encodes apolypeptide comprising (i) a sequence having greater than 43% identityto the amino acid sequence of SEQUENCE ID NO:2; (ii) a sequencecomprising at least about 10 contiguous amino acids that have greaterthan 43% identity to 10 contiguous amino acids of SEQUENCE ID NO:2, or acomplement or reverse complement of said polynucleotide. In certainembodiments, the polynucleotide will have at least 70% identity to theDWF4 polypeptide-coding region of SEQ ID NO: 1 or to complements andreverse complements of this region. In further embodiments, the isolateddwf4 polynucleotide comprises the nucleotide sequence of SEQ ID NO:1,complements and reverse complements thereof. The polynucleotide may alsocomprise at least 30 consecutive nucleotides of SEQ ID NO: 1.

[0018] In another aspect, the invention includes an isolated dwf4polynucleotide comprising (i) a sequence having at least 50% identity toSEQ ID NO: 1, complements and reverse complements thereof or (ii) asequence comprising at least about 15 contiguous nucleotides that has atleast 50% identity to SEQ ID NO: 1, complements and reverse complementsthereof. In certain embodiments, the isolated dwf4 polynucleotide has atleast 50% identity to the DWF4 polypeptide-coding region of SEQ ID NO:1, complements and reverse complements thereof. In further embodiments,the isolated dwf4 polynucleotides described herein comprise thenucleotide sequence of SEQ ID NO:1, complements and reverse complementsthereof or nucleotide sequences comprising at least 30 consecutivenucleotides of SEQ ID NO:1. Any of the dwf4 polynucleotides describedherein may be genomic DNA and may include introns. Further, in otherembodiments, the dwf4 polynucleotide includes a dwf4 control controlelement comprising a polynucleotide selected from the group consistingof (i) a sequence having at least 50% identity to nucleotides 1 to 3202of SEQ ID NO:1; (ii) a fragment of (i) which includes a dwf4 controlelement; and (iii) complements and reverse complements of (i) or (ii).In still further embodiments, the polynucleotide includes a dwf4 controlelement comprising a polynucleotide selected from the group consistingof (i) a sequence having at least 50% identity to nucleotides 6111 to6468 corresponding to the 3′ UTR of SEQ ID NO:1; (ii) a fragment of (i)which includes a dwf4 3′ UTR; and (iii) complements and reversecomplements of (i) or (ii). In certain embodiments, the polynucleotideincludes a dwf4 polynucleotide selected from the group consisting of (i)a sequence having at least 50% identity to the sequences correspondingto the introns of SEQ ID NO:1; (ii) a fragment of (i) which includes adwf4 intro; and (iii) complements and reverse complements of (i) and(ii). Introns are found, for example, in the following regions:nucleotides 3424 to 3503 of SEQ ID NO:1; nucleotides 3829 to 3913 of SEQID NO:1; nucleotides 4067 to 4164 of SEQ ID NO:1; nucleotides 4480 to4531 of SEQ ID NO:1; nucleotides 4725 to 4815 of SEQ ID NO:1;nucleotides 4895 to 5000 of SEQ ID NO:1; and nucleotides 5111 to 5864 ofSEQ ID NO:1. 54. In still further embodiments, any of thepolynucleotides described herein can operably linked to a nucleic acidmolecule encoding a heterologous polypeptide (e.g., a cytochrome P450polypeptide), for example, as a chimeric polynucleotide.

[0019] In another aspect, the invention includes recombinant vectorscomprising (i) one or more of the polynucleotides described above; and(ii) control elements operably linked to the one or morepolynucleotides, whereby a coding sequence within said polynucleotidecan be transcribed and translated in a host cell. In certainembodiments, the recombinant vector comprises (a) any of thepolynucleotides which include a dwf4 control element described above(e.g., promoter or intron); and (b) a nucleic acid molecule comprising acoding sequence operably linked to the dwf4 control element.

[0020] Host cells comprising and/or transformed with any of therecombinant vectors described herein are also provided. In certainembodiments, the host cells are cultured ex vivo while in otherembodiments, the dwf4 polynucleotide is provided the host cell in vivo.In certain embodiments the DWF4 polypeptide is provided in amounts suchthat a plant is regenerated.

[0021] In another aspect, the present invention includes a method ofmodulating a DWF4 polypeptide comprising the following steps: (a)providing a host cell as described herein; and (b) culturing said hostcell under conditions whereby the dwf4 polynucleotide included in thehost cell is transcribed. In certain embodiments, the dwf4polynucleotide is overexpressed. Alternatively, in other embodiments,the polynucleotide included in the host cell inhibits expression ofdwf4.

[0022] In yet another aspect, the present invention includes atransgenic plant comprising any of the recombinant vectors describedherein.

[0023] In yet another aspect, the invention includes a method ofproducing a recombinant polypeptide comprising the following steps: (a)providing a host cell as described herein; and (b) culturing said hostcell under conditions whereby the recombinant polypeptide encoded by thecoding sequence present in said recombinant vector is expressed.

[0024] In a still further aspect, the invention includes a method ofproducing a transgenic plant comprising the steps of (a) introducing apolynucleotide described herein into a plant cell to produce atransformed plant cell; and (b) producing a transgenic plant from thetransformed plant cell.

[0025] Methods for producing a transgenic plant having an alteredphenotype relative to the wild-type plant comprising the followingsteps: introducing at least one polynucleotide described herein into aplant cell; and producing a transgenic plant from the plant cell, saidtransgenic plant having an altered phenotype relative to the wild-typeplant are also included in the present invention. The altered phenotypeincludes altered morphological appearance and altered biochemicalactivity, for example, altered (reduced or increased) cell length in anycell or tissue, altered (extended or decreased) periods of flowering,altered (increased or decreased) branching, altered (increased ordecreased) seed production, altered (increased or decreased) leaf size,altered (elongated or shortened) hypocotyls, altered (increased ordecreased) plant height, altered heme-thiolate enzyme activity, alteredmonooxygenase activity, altered 22α-hydroxylase activity, regulation ofbrassinosteriod synthesis, regulation of gibberellic acid, regulation ofcytokinins, regulation of auxins, altered resistance to plant pathogens,altered growth at low temperatures, altered growth in dark conditionsand altered sterol composition. In certain embodiments, the at least onepolynucleotide is operably linked to a promoter selected from the groupconsisting of a tissue-specific promoter, an inducible promoter or aconstitutive promoter. The polynucleotide can be overexpressed or it caninhibit expression of dwf4. In a still further embodiment, at least twopolynucleotides are introduced into the plant cell. Each polynucleotideis operably linked to a different tissue-specific promoter such that onepolynucleotide is overexpressed while the other inhibits expression ofdwf4.

[0026] In yet another aspect, the invention includes a method foraltering the biochemical activity of a cell comprising the followingsteps: introducing at least one polynucleotide described herein; andculturing the cell under conditions such that the biochemical activityof the cell is altered. Biochemical activity includes, for example,altered heme-thiolate enzyme activity, altered monooxygenase activity,altered 22α-hydroxylase activity, regulation of gibberellic acid,regulation of cytokinins, regulation of auxins, and altered sterolcomposition. In certain embodiments, the cell is cultured ex vivo. Inother embodiments, the dwf4 polynucleotide is provided to the cell invivo. In still other embodiments, more than one dwf4 polynucleotides areprovided to the cell.

[0027] In yet another aspect, the invention includes a method forregulating the cell cycle of a plant cell comprising the following stepsproviding a dwf4 polynucleotide to a plant cell; and expressing the dwf4polynucleotide to provide a DWF4 polypeptide, wherein the DWF4polypeptide is provided in amounts such that cell cycling is regulated.In certain embodiments, the plant cell is provided in vitro and iscultured under conditions suitable for providing the DWF4 polypeptide.In still other embodiments, the dwf4 polynucleotide is provided in vivo.

[0028] In yet another aspect, the invention includes an isolated DWF4polypeptide comprising (i) a sequence having greater than 43% identityto SEQ ID NO:2 or (ii) fragments of (i) that confer a DWF4 phenotypewhen expressed in a host organism. In certain embodiments, the isolatedDWF4 polypeptide comprises the amino acid sequence of SEQ ID NO:2. Incertain embodiments, the invention includes a chimeric polypeptidecomprising a DWF4 polypeptide as described herein and a heterologouspolypeptide, for example a cytochrome P450 polypeptide.

[0029] Any of the polynucleotides or polypeptides described herein canbe used in diagnostic assays; to generate antibodies. Further, theantibodies and fragments thereof can also be used in diagnostic assays,to produce immunogenic compositions or the like.

[0030] These and other objects, aspects, embodiments and advantages ofthe present invention will readily occur to those of ordinary skill inthe art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031]FIG. 1 depicts a proposed biosynthetic pathway for BL. CR goesthrough at least two different pathways, referred to as the early C-6oxidation (right column) and late C-6 oxidation (left column) pathways.Steps mediated by DWF4, CPD (Szerkeres et al. (1996), supra), DET2(Fujioka and Skaurai (1997a), infra; Li et al. (1997), supra) and LKB(Yokota et al. (1997), infra) are indicated.

[0032]FIGS. 2A and B depict schematic representations of the DWF4 geneand protein. FIG. 2A depicts the DWF4 coding sequence (1542 bp) andshows that the coding sequence contains eight exons and seven introns.The exons and introns range in length from 93 to 604 and 84 to 754 bp,respectively. All of the introns are bordered by typical consensussplice junctions, 5′-GU and AG-3′. Closed rectangles indicate exons. TheT-DNA position in dwf4-1 is marked with an arrow. FIG. 2B shows therelative positions of the major domains in DWF4 cytochrome P450. All ofthe major domains found in the cytochrome P450 superfamily are conservedin DWF4. The estimated molecular mass and isoelectric point of the DWF4protein were 58 kD and 7.28, respectively. Hydropathy plotting andprotein localization prediction by the PSORT software package (Nakai andKaneshia (1992) Genomics 14:897-911) suggested that the protein mayreside in a membrane of the endoplasmic reticulum as an integralprotein. Mutations identified in the other dwf4 alleles are indicated.

[0033]FIG. 3 depicts alignment of cytochrome P450 proteins thatexhibited the most similarity to DWF4 in BLAST searches. GenBankaccession numbers are AF044216 (DWF4; CYP90B), X87368 (CPD; CYP90A),U54770 (tomato; CYP85), D64003 (cyanobacteria; CYP120), U3259 (maize;CYP88), U68234 (zebrafish; CYP26), and M13785 (human; CYP3A3X). Dashesindicate gaps introduced to maximize alignment. Domains indicated inFIG. 2B are highlighted in a box. Amino acid residues that areconserved >50% between the compared sequences are highlighted by areverse font, and identical residues between DWF4 and CPD are boxed anditalicized. Open triangles are placed under the 100% conserved residues.Closed triangles locate functionally important amino acid residues, forexample, threonine (T) at 369, which is thought to bind molecularoxygen, and cysteine (C) at 516, which links to a heme prosthetic groupby a thiolate bond. X's indicate mutated residues in dwf4 alleles.Multiple sequence alignment was performed using PILEUP in the GeneticsComputer Group package, and box shading was made possible by theALSCRIPT package (Barton (1993) Protein Eng. 6:37-40).

[0034]FIG. 4 depicts the phylogenetic Relationship between DWF4 andSelected Cytochrome P450s. DWF4 did not cluster with the group A plantcytochrome P450s that are known to mediate plant-specific reactions(Durst and Nelson 1995). CYP90A, CYP85, and DWF4, which are thought tobe involved in BR metabolism, branched from CYP88, which mediates GAbiosynthesis. GenBank accession numbers for the group A cytochrome P450sare M32885 (avocado; CYP71A1), P48421 (Arabidopsis; CYP83), P48418(petunia; CYP75A1), and X71658 (eggplant; CYP76A1). The DISTANCE utilityin the Genetics Computer Group software package was employed tocalculate the relationships.

[0035]FIG. 5 depicts a comparison of wild-type and dwf4 hypocotyl growthrates. Circles indicate wild-type and square indicate dwf4. Each datapoint represents the average of 10 seedlings.

[0036]FIG. 6 depicts responses to cell elongation signals. BLmeasurements were performed with dwf4-3 and the corresponding wild-typecontrol, Enkheim. Open bars indicate the wild type. Filled bars indicatedwf4. Lines above the bars represent one standard deviation. On thehorizontal axis, “light” refers to light-grown controls; “dark” refersto dark-grown controls; “hy2” refers to DWF4 and dwf4 plants in a hy2background; “GA” refers to plants grown in 10⁻⁵ M GA; “2,4-D” refers toplants grown in 10⁻⁸ M 2,4-D; “−BR” refers to liquid-grown controls; and“+BR” refers to liquid-grown controls with 1108 M BL.

[0037]FIG. 7 depicts pedicel elongation of dwf4 mature plants inresponse to exogenous application of BR. Measurements were performedwith the BR-fed plants. dwf4-1 plants were more sensitive tointermediates belonging to the late C-6 oxidation pathway (10⁻¹⁶ M6-deoxoCT and 10⁻¹⁶ M 6-deoxoTE) compared with compounds in the earlyC-6 pathway (10⁻⁵ M CT and 10⁻⁵ M TE). BL (10⁻⁷ M) induced almost thesame amount of elongation with one-tenth the concentration of itsprecursors. Rescue by 22-OHCR (10⁻⁵ M), which is structurally similar tothe presumed precursor CR, except for a 22α-hydroxyl functional group,shows that the only defect in dwf4 is the C-22 hydroxylation reaction.Complementing intermediates and BL induced dramatic elongation in theelongating zone of the inflorescence and pedicel, but fertility was notincreased. Data represent the means±SE of 15 to 20 pedicels. “CTRL”refers to control; “WT” refers to wild type.

[0038]FIG. 8 depicts the increase in inflorescence growth of threetransformants which overexpress dwf4 as compared to wild type (Ws-2).The length of inflorescences of DWF4 overexpression lines increased morethan 20% compared to that of wild type. The length of the plant wasmeasured at maturity. Each date point is a mean value of more than 9plants, except AOD4-60 which represents 2 plants.

[0039]FIG. 9 depicts the increase in seed production of threetransformants which overexpress dwf4 as compared to wild type (Ws-2).Seeds were harvested from individual plants of each genotype (n>5).Seeds from each plant were weighed and a mean value calculated. TheFigure shows percent increase over wild type.

[0040] FIGS. 10(A)-10(G) depict the nucleotide sequence of wild-typedwf4 (SEQ ID NO:1, see, also, GenBank Accession Number AF044216). Thedwf4 polynucleotide includes a coding region between nucleotides 3203and 6110, inclusive. The coding region includes the following eightexons: nucleotides 3203 to 3423, inclusive; nucleotides 3504 to 3828,inclusive; nucleotides 3914 to 4066, inclusive; nucleotides 4165 to4479, inclusive; nucleotides 4632 to 4724, inclusive; nucleotides 4816to 4894, inclusive; nucleotides 5001 to 5110, inclusive and nucleotides5865 to 6110, inclusive. The exons are indicated by a bar beneath thenucleotide sequence. A 5′ control region (e.g., promoter) extends fromnucleotides 1 to 3202. A 3′ untranslated region (UTR), corresponds tothe region extending from nucleotide to 6011 to approximately nucleotide6468 of FIG. 10 (SEQ ID NO:1) and a TATA signal extending approximatelyfrom nucleotides 3060 to 3125. As described in the Examples, mutantalleles of dwf4 have also been characterized. For example, dwf4-1contains an approximately 20 kb insert between nucleotides 5202 and5203. dwf4-2 has a 9 base pair deletion corresponding to amino acids324-326. In mutant allele dwf4-3, the guanine (G) residue at position4332 is replaced with an adenine (A) residue to create a premature stopcodon and truncate the DWF4 protein at amino acid 289.

[0041]FIG. 11 depicts the amino acid sequence of the DWF4 polypeptide(GenBank Accession Number AAC05093, SEQ ID NO:2). The polypeptide is 513amino acids in length.

[0042]FIG. 12 depicts seedling phenotypes of twelve-day-old dwf4-1, wildtype, epi-BL-treated wild type, and AOD4 lines grown in the light anddark, particularly quantification of hypocotyl and root growth. Theaverage lengths of 16 seedlings are displayed with the standarddeviation. Increased BR concentration supplied exogenously orendogenously resulted in both elongated hypocotyls and shortened roots.

DETAILED DESCRIPTION

[0043] The novel dwf4 polynucleotides and DWF4 polypeptides describedherein are important molecules in regulating cell growth and sterolsynthesis. The present inventors have shown that dwf4 encodes acytochrome P450 monooxygenase having 43% sequence identity to theprotein termed Constitutive Phoromorphogenesis and Dwfarism (CPD). Asshown in FIG. 1, both CPD and DWF4 polypeptides appear to regulatebiosynthesis of brassinosteriods, for example brassinolide (BL).However, unlike previously characterized proteins (e.g, CPD), DWF4appears to act as a “gatekeeper” in these biosynthetic pathways in thatits substrates (e.g., 6-Oxo campestanol and 6α-Hydroxy campestanol) areapproximately 500 times more prevalent than the downstream molecules.Thus, the present invention represents an important discovery inunderstanding and regulating cell growth.

[0044] Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified molecules or process parameters as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments of the inventiononly, and is not intended to be limiting. In addition, the practice ofthe present invention will employ, unless otherwise indicated,conventional methods of plant biology, virology, microbiology, molecularbiology, recombinant DNA techniques and immunology all of which arewithin the ordinary skill of the art. Such techniques are explainedfully in the literature. See, e.g., Evans, et al., Handbook of PlantCell Culture (1983, Macmillan Publishing Co.); Binding, Regeneration ofPlants, Plant Protoplasts (1985, CRC Press); Sambrook, et al., MolecularCloning: A Laboratory Manual (2nd Edition, 1989); DNA Cloning: APractical Approach, vol. I & II (D. Glover, ed.); OligonucleotideSynthesis (N. Gait, ed., 1984); A Practical Guide to Molecular Cloning(1984); and Fundamental Virology, 2nd Edition, vol. I & II (B. N. Fieldsand D. M. Knipe, eds.).

[0045] All publications, patents and patent applications cited herein,whether supra or infra, are hereby incorporated by reference in theirentirety.

[0046] It must be noted that, as used in this specification and theappended claims, the singular forms “a”, “an” and “the” include pluralreferents unless the content clearly dictates otherwise. Thus, forexample, reference to “a polypeptide” includes a mixture of two or morepolypeptides, and the like.

[0047] The following amino acid abbreviations are used throughout thetext: Alanine: Ala (A) Arginine: Arg (R) Asparagine: Asn (N) Asparticacid: Asp (D) Cysteine: Cys (C) Glutamine: Gln (Q) Glutamic acid: Glu(E) Glycine: Gly (G) Histidine: His (H) Isoleucine: Ile (I) Leucine: Leu(L) Lysine: Lys (K) Methionine: Met (M) Phenylalanine: Phe (F) Proline:Pro (P) Serine: Ser (S) Threonine: Thr (T) Tryptophan: Trp (W) Tyrosine:Tyr (Y) Valine: Val (V)

[0048] Definitions

[0049] In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

[0050] The terms “nucleic acid molecule” and “polynucleotide” are usedinterchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides, or analogsthereof. This term refers only to the primary structure of the moleculeand thus includes double- and single-stranded DNA and RNA. It alsoincludes known types of modifications, for example, labels which areknown in the art, methylation, “caps”, substitution of one or more ofthe naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example proteins (including e.g., nucleases, toxins, antibodies,signal peptides, poly-L-lysine, etc.), those with intercalators (e.g.,acridine, psoralen, etc.), those containing chelates (e.g., metals,radioactive metals, boron, oxidative metals, etc.), those containingalkylators, those with modified linkages (e.g., alpha anomeric nucleicacids, etc.), as well as unmodified forms of the polynucleotide.Polynucleotides may have any three-dimensional structure, and mayperform any function, known or unknown. Nonlimiting examples ofpolynucleotides include a gene, a gene fragment, exons, introns,messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers.

[0051] A polynucleotide is typically composed of a specific sequence offour nucleotide bases: adenine (A); cytosine (C); guanine (G); andthymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA).Thus, the term polynucleotide sequence is the alphabeticalrepresentation of a polynucleotide molecule. This alphabeticalrepresentation can be input into databases in a computer having acentral processing unit and used for bioinformatics applications such asfunctional genomics and homology searching.

[0052] Techniques for determining nucleic acid and amino acid “sequenceidentity” are known in the art. Typically, such techniques includedetermining the nucleotide sequence of the mRNA for a gene and/ordetermining the amino acid sequence encoded thereby, and comparing thesesequences to a second nucleotide or amino acid sequence. In general,“identity” refers to an exact nucleotide-to-nucleotide or aminoacid-to-amino acid correspondence of two polynucleotides or polypeptidesequences, respectively. Two or more sequences (polynucleotide or aminoacid) can be compared by determining their “percent identity.” Thepercent identity of two sequences, whether nucleic acid or amino acidsequences, is the number of exact matches between two aligned sequencesdivided by the length of the shorter sequences and multiplied by 100. Anapproximate alignment for nucleic acid sequences is provided by thelocal homology algorithm of Smith and Waterman, Advances in AppliedMathematics 2:482-489 (1981). This algorithm can be applied to aminoacid sequences by using the scoring matrix developed by Dayhoff, Atlasof Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl.3:353-358, National Biomedical Research Foundation, Washington, D.C.,USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763(1986). An exemplary implementation of this algorithm to determinepercent identity of a sequence is provided by the Genetics ComputerGroup (Madison, Wis.) in the “BestFit” utility application. The defaultparameters for this method are described in the Wisconsin SequenceAnalysis Package Program Manual, Version 8 (1995) (available fromGenetics Computer Group, Madison, Wis.). A preferred method ofestablishing percent identity in the context of the present invention isto use the MPSRCH package of programs copyrighted by the University ofEdinburgh, developed by John F. Collins and Shane S. Sturrok, anddistributed by IntelliGenetics, Inc. (Mountain View, Calif.). From thissuite of packages the Smith-Waterman algorithm can be employed wheredefault parameters are used for the scoring table (for example, gap openpenalty of 12, gap extension penalty of one, and a gap of six). From thedata generated the “Match” value reflects “sequence identity.” Othersuitable programs for calculating the percent identity or similaritybetween sequences are generally known in the art, for example, anotheralignment program is BLAST, used with default parameters. For example,BLASTN and BLASTP can be used using the following default parameters:genetic code=standard; filter=none; strand=both; cutoff=60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+Swiss protein+Spupdate+PIR. Details of these programs canbe found at the following internet address:http://www.ncbi.nlm.gov/cgi-bin/BLAST.

[0053] Alternatively, the degree of sequence similarity betweenpolynucleotides can be determined by hybridization of polynucleotidesunder conditions that form stable duplexes between homologous regions,followed by digestion with single-stranded-specific nuclease(s), andsize determination of the digested fragments. Two DNA, or twopolypeptide sequences are “substantially homologous” to each other whenthe sequences exhibit at least about 43%-60%, preferably 60-70%, morepreferably 70%-85%, more preferably at least about 85%-90%, morepreferably at least about 90%-95%, and most preferably at least about95%-98% sequence identity over a defined length of the molecules, or anypercentage between the above-specified ranges, as determined using themethods above. As used herein, substantially homologous also refers tosequences showing complete identity to the specified DNA or polypeptidesequence. DNA sequences that are substantially homologous can beidentified in a Southern hybridization experiment under, for example,stringent conditions, as defined for that particular system. Definingappropriate hybridization conditions is within the skill of the art.See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic AcidHybridization, supra.

[0054] The degree of sequence identity between two nucleic acidmolecules affects the efficiency and strength of hybridization eventsbetween such molecules. A partially identical nucleic acid sequence willat least partially inhibit a completely identical sequence fromhybridizing to a target molecule. Inhibition of hybridization of thecompletely identical sequence can be assessed using hybridization assaysthat are well known in the art (e.g., Southern blot, Northern blot,solution hybridization, or the like, see Sambrook, et al., MolecularCloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor,N.Y.). Such assays can be conducted using varying degrees ofselectivity, for example, using conditions varying from low to highstringency. If conditions of low stringency are employed, the absence ofnon-specific binding can be assessed using a secondary probe that lackseven a partial degree of sequence identity (for example, a probe havingless than about 30% sequence identity with the target molecule), suchthat, in the absence of non-specific binding events, the secondary probewill not hybridize to the target.

[0055] When utilizing a hybridization-based detection system, a nucleicacid probe is chosen that is complementary to a target nucleic acidsequence, and then by selection of appropriate conditions the probe andthe target sequence “selectively hybridize,” or bind, to each other toform a hybrid molecule. A nucleic acid molecule that is capable ofhybridizing selectively to a target sequence under “moderatelystringent” typically hybridizes under conditions that allow detection ofa target nucleic acid sequence of at least about 10⁻¹⁴ nucleotides inlength having at least approximately 70% sequence identity with thesequence of the selected nucleic acid probe. Stringent hybridizationconditions typically allow detection of target nucleic acid sequences ofat least about 10⁻¹⁴ nucleotides in length having a sequence identity ofgreater than about 90-95% with the sequence of the selected nucleic acidprobe. Hybridization conditions useful for probe/target hybridizationwhere the probe and target have a specific degree of sequence identity,can be determined as is known in the art (see, for example, Nucleic AcidHybridization: A Practical Approach, editors B. D. Hames and S. J.Higgins, (1985) Oxford; Washington, D.C.; IRL Press).

[0056] With respect to stringency conditions for hybridization, it iswell known in the art that numerous equivalent conditions can beemployed to establish a particular stringency by varying, for example,the following factors: the length and nature of probe and targetsequences, base composition of the various sequences, concentrations ofsalts and other hybridization solution components, the presence orabsence of blocking agents in the hybridization solutions (e.g.,formamide, dextran sulfate, and polyethylene glycol), hybridizationreaction temperature and time parameters, as well as, varying washconditions. The selection of a particular set of hybridizationconditions is selected following standard methods in the art (see, forexample, Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, (1989) Cold Spring Harbor, N.Y.).

[0057] A “gene” as used in the context of the present invention is asequence of nucleotides in a genetic nucleic acid (chromosome, plasmid,etc.) with which a genetic function is associated. A gene is ahereditary unit, for example of an organism, comprising a polynucleotidesequence that occupies a specific physical location (a “gene locus” or“genetic locus”) within the genome of an organism. A gene can encode anexpressed product, such as a polypeptide or a polynucleotide (e.g.,tRNA). Alternatively, a gene may define a genomic location for aparticular event/function, such as the binding of proteins and/ornucleic acids, wherein the gene does not encode an expressed product.Typically, a gene includes coding sequences, such as, polypeptideencoding sequences, and non-coding sequences, such as, promotersequences, polyadenlyation sequences, transcriptional regulatorysequences (e.g., enhancer sequences). Many eucaryotic genes have “exons”(coding sequences) interrupted by “introns” (non-coding sequences). Incertain cases, a gene may share sequences with another gene(s) (e.g.,overlapping genes).

[0058] A “coding sequence” or a sequence which “encodes” a selectedpolypeptide, is a nucleic acid molecule which is transcribed (in thecase of DNA) and translated (in the case of mRNA) into a polypeptide,for example, in vivo when placed under the control of appropriateregulatory sequences (or “control elements”). The boundaries of thecoding sequence are typically determined by a start codon at the 5′(amino) terminus and a translation stop codon at the 3′ (carboxy)terminus. A coding sequence can include, but is not limited to, cDNAfrom viral, procaryotic or eucaryotic mRNA, genomic DNA sequences fromviral or procaryotic DNA, and even synthetic DNA sequences. Atranscription termination sequence may be located 3′ to the codingsequence. Other “control elements” may also be associated with a codingsequence. A DNA sequence encoding a polypeptide can be optimized forexpression in a selected cell by using the codons preferred by theselected cell to represent the DNA copy of the desired polypeptidecoding sequence. “Encoded by” refers to a nucleic acid sequence whichcodes for a polypeptide sequence, wherein the polypeptide sequence or aportion thereof contains an amino acid sequence of at least 3 to 5 aminoacids, more preferably at least 8 to 10 amino acids, and even morepreferably at least 15 to 20 amino acids from a polypeptide encoded bythe nucleic acid sequence. Also encompassed are polypeptide sequenceswhich are immunologically identifiable with a polypeptide encoded by thesequence.

[0059] Typical “control elements”, include, but are not limited to,transcription promoters, transcription enhancer elements, transcriptiontermination signals, polyadenylation sequences (located 3′ to thetranslation stop codon), sequences for optimization of initiation oftranslation (located 5′ to the coding sequence), translation enhancingsequences, and translation termination sequences. Transcriptionpromoters can include inducible promoters (where expression of apolynucleotide sequence operably linked to the promoter is induced by ananalyte, cofactor, regulatory protein, etc.), tissue-specific promoters(where expression of a polynucleotide sequence operably linked to thepromoter is induced only in selected tissue), repressible promoters(where expression of a polynucleotide sequence operably linked to thepromoter is induced by an analyte, cofactor, regulatory protein, etc.),and constitutive promoters.

[0060] A control element, such as a promoter, “directs thetranscription” of a coding sequence in a cell when RNA polymerase willbind the promoter and transcribe the coding sequence into mRNA, which isthen translated into the polypeptide encoded by the coding sequence.

[0061] “Expression enhancing sequences” typically refer to controlelements that improve transcription or translation of a polynucleotiderelative to the expression level in the absence of such control elements(for example, promoters, promoter enhancers, enhancer elements, andtranslational enhancers (e.g., Shine and Delagarno sequences).

[0062] “Operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences. The control elements need not be contiguous with the codingsequence, so long as they function to direct the expression thereof.Thus, for example, intervening untranslated yet transcribed sequencescan be present between a promoter and the coding sequence and thepromoter can still be considered “operably linked” to the codingsequence.

[0063] A “heterologous sequence” as used herein typically refers to anucleic acid sequence that is not normally found in the cell or organismof interest. For example, a DNA sequence encoding a polypeptide can beobtained from a plant cell and introduced into a bacterial cell. In thiscase the plant DNA sequence is “heterologous” to the native DNA of thebacterial cell.

[0064] The “native sequence” or “wild-type sequence” of a gene is thepolynucleotide sequence that comprises the genetic locus correspondingto the gene, e.g., all regulatory and open-reading frame codingsequences required for expression of a completely functional geneproduct as they are present in the wild-type genome of an organism. Thenative sequence of a gene can include, for example, transcriptionalpromoter sequences, translation enhancing sequences, introns, exons, andpoly-A processing signal sites. It is noted that in the generalpopulation, wild-type genes may include multiple prevalent versions thatcontain alterations in sequence relative to each other and yet do notcause a discernible pathological effect. These variations are designated“polymorphisms” or “allelic variations.”

[0065] “Recombinant” as used herein to describe a nucleic acid moleculemeans a polynucleotide of genomic, cDNA, semisynthetic, or syntheticorigin which, by virtue of its origin or manipulation: (1) is notassociated with all or a portion of the polynucleotide with which it isassociated in nature; and/or (2) is linked to a polynucleotide otherthan that to which it is linked in nature. The term “recombinant” asused with respect to a protein or polypeptide means a polypeptideproduced by expression of a recombinant polynucleotide.

[0066] By “vector” is meant any genetic element, such as a plasmid,phage, transposon, cosmid, chromosome, virus etc., which is capable oftransferring gene sequences to target cells. Generally, a vector iscapable of replication when associated with the proper control elements.Thus, the term includes cloning and expression vehicles, as well asviral vectors and integrating vectors.

[0067] As used herein, the term “expression cassette” refers to amolecule comprising at least one coding sequence operably linked to acontrol sequence which includes all nucleotide sequences required forthe transcription of cloned copies of the coding sequence and thetranslation of the mRNAs in an appropriate host cell. Such expressioncassettes can be used to express eukaryotic genes in a variety of hostssuch as bacteria, blue-green algae, plant cells, yeast cells, insectcells and animal cells. Under the invention, expression cassettes caninclude, but are not limited to, cloning vectors, specifically designedplasmids, viruses or virus particles. The cassettes may further includean origin of replication for autonomous replication in host cells,selectable markers, various restriction sites, a potential for high copynumber and strong promoters.

[0068] A cell has been “transformed” by an exogenous polynucleotide whenthe polynucleotide has been introduced inside the cell. The exogenouspolynucleQtide may or may not be integrated (covalently linked) intochromosomal DNA making up the genome of the cell. In prokaryotes andyeasts, for example, the exogenous DNA may be maintained on an episomalelement, such as a plasmid. With respect to eucaryotic cells, a stablytransformed cell is one in which the exogenous DNA has become integratedinto the chromosome so that it is inherited by daughter cells throughchromosome replication. This stability is demonstrated by the ability ofthe eucaryotic cell to establish cell lines or clones comprised of apopulation of daughter cells containing the exogenous DNA.

[0069] “Recombinant host cells,” “host cells,” “cells,” “cell lines,”“cell cultures,” and other such terms denoting procaryoticmicroorganisms or eucaryotic cell lines cultured as unicellularentities, are used interchangeably, and refer to cells which can be, orhave been, used as recipients for recombinant vectors or other transferDNA, and include the progeny of the original cell which has beentransfected. It is understood that the progeny of a single parental cellmay not necessarily be completely identical in morphology or in genomicor total DNA complement to the original parent, due to accidental ordeliberate mutation. Progeny of the parental cell which are sufficientlysimilar to the parent to be characterized by the relevant property, suchas the presence of a nucleotide sequence encoding a desired peptide, areincluded in the progeny intended by this definition, and are covered bythe above terms.

[0070] The term “dwf4 polynucleotide” refers to a polynucleotide derivedfrom the dwf4 gene. The gene encodes the protein referred to herein asDWF4. DWF4 is a cytochrome P450 cytochrome P450 that mediates multiple22α-hydroxylation steps in brassinosteroid biosynthesis (see, FIG. 1).The dwf4 polynucleotide sequence and corresponding amino acid sequenceare shown in FIGS. 10 and 11 (SEQ ID NO:1, SEQ ID NO:2 and GenBankaccession No. AF044216). As shown in FIG. 10, the dwf4 coding sequencespans the region from nucleotide positions 3203 to 6110 and the upstream5′ UTR, including the promoter region, spans nucleotide positions 1 to3202. A functional 1.1 kb control element is also described in theExamples. A 3′ UTR spans nucleotide positions 6111 to approximately 6468of SEQ ID NO:1. The term as used herein encompasses a polynucleotideincluding a native sequence depicted in FIG. 10, as well asmodifications and fragments thereof.

[0071] The term encompasses alterations to the polynucleotide sequence,so long as the alteration results in a plant displaying one or more dwf4phenotypic traits (described below) when the polynucleotide is expressedin a plant. Such modifications typically include deletions, additionsand substitutions, to the native dwf4 sequence, so long as the mutationresults in a plant displaying a dwf4 phenotype as defined below. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through mutations of plants which expressthe dwf4 polynucleotide or errors due to PCR amplification. The termencompasses expressed allelic variants of the wild-type dwf4 sequencewhich may occur by normal genetic variation or are produced by geneticengineering methods and which result in a detectable change in thewild-type dwf4 phenotype.

[0072] The term “dwf4 phenotype” as used herein refers to anymicroscopic or macroscopic change in structure or morphology of a plant,such as a transgenic plant, as well as biochemical differences, whichare characteristic of a dwf4 plant, compared to a progenitor, wild-typeplant cultivated under the same conditions. Generally, morphologicaldifferences include multiple short stems, short rounded leaves, loss offertility due to reduced stamen length, and delayed development.Dark-grown dwf4 seedlings possess short hypocotyls, open cotyledons, anddeveloping leaves. The height of such plants will typically be 75% orless of the wild-type plant, more typically 50% or less of the wild-typeplant, and even more typically 25% or less of the wild-type plant, orany integer in between. Additional phenotypic morphological attributesof the dwf4 mutant are summarized in Table 1 of the examples.Biochemically, dwf4 hypocotyls are converted to wild-type length withthe application of BL.

[0073] A “polypeptide” is used in it broadest sense to refer to acompound of two or more subunit amino acids, amino acid analogs, orother peptidomimetics. The subunits may be linked by peptide bonds or byother bonds, for example ester, ether, etc. As used herein, the term“amino acid” refers to either natural and/or unnatural or syntheticamino acids, including glycine and both the D or L optical isomers, andamino acid analogs and peptidomimetics. A peptide of three or more aminoacids is commonly called an oligopeptide if the peptide chain is short.If the peptide chain is long, the peptide is typically called apolypeptide or a protein. Full-length proteins, analogs, mutants andfragments thereof are encompassed by the definition. The terms alsoinclude postexpression modifications of the polypeptide, for example,glycosylation, acetylation, phosphorylation and the like. Furthermore,as ionizable amino and carboxyl groups are present in the molecule, aparticular polypeptide may be obtained as an acidic or basic salt, or inneutral form. A polypeptide may be obtained directly from the sourceorganism, or may be recombinantly or synthetically produced (see furtherbelow).

[0074] A “DWF4” polypeptide is a polypeptide as defined above, which isderived from a 22α-hydroxylase that functions in the brassinolide (BL)biosynthetic pathway (see, FIG. 1). The native sequence of full-lengthDWF4 is shown in FIG. 11 (SEQ ID NO:2). However, the term encompassesmutants and fragments of the native sequence so long as the proteinfunctions for its intended purpose.

[0075] The term “DWF4 analog” refers to derivatives of DWF4, orfragments of such derivatives, that retain desired function, e.g., asmeasured in assays as described further below. In general, the term“analog” refers to compounds having a native polypeptide sequence andstructure with one or more amino acid additions, substitutions(generally conservative in nature) and/or deletions, relative to thenative molecule, so long as the modifications do not destroy desiredactivity. Preferably, the analog has at least the same activity as thenative molecule. Methods for making polypeptide analogs are known in theart and are described further below.

[0076] Particularly preferred analogs include substitutions that areconservative in nature, i.e., those substitutions that take place withina family of amino acids that are related in their side chains.Specifically, amino acids are generally divided into four families: (1)acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine;(3) non-polar—alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine,asparagine, glutamine, cysteine, serine threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified asaromatic amino acids. For example, it is reasonably predictable that anisolated replacement of leucine with isoleucine or valine, an aspartatewith a glutamate, a threonine with a serine, or a similar conservativereplacement of an amino acid with a structurally related amino acid,will not have a major effect on the biological activity. It is to beunderstood that the terms include the various sequence polymorphismsthat exist, wherein amino acid substitutions in the protein sequence donot affect the essential functions of the protein.

[0077] By “purified” and “isolated” is meant, when referring to apolypeptide or polynucleotide, that the molecule is separate anddiscrete from the whole organism with which the molecule is found innature; or devoid, in whole or part, of sequences normally associatedwith it in nature; or a sequence, as it exists in nature, but havingheterologous sequences (as defined below) in association therewith. Itis to be understood that the term “isolated” with reference to apolynucleotide intends that the polynucleotide is separate and discretefrom the chromosome from which the polynucleotide may derive. The term“purified” as used herein preferably means at least 75% by weight, morepreferably at least 85% by weight, more preferably still at least 95% byweight, and most preferably at least 98% by weight, of biologicalmacromolecules of the same type are present. An “isolated polynucleotidewhich encodes a particular polypeptide” refers to a nucleic acidmolecule which is substantially free of other nucleic acid moleculesthat do not encode the subject polypeptide; however, the molecule mayinclude some additional bases or moieties which do not deleteriouslyaffect the basic characteristics of the composition.

[0078] By “fragment” is intended a polypeptide or polynucleotideconsisting of only a part of the intact sequence and structure of thereference polypeptide or polynucleotide, respectively. The fragment caninclude a 3′ or C-terminal deletion or a 5′ or N-terminal deletion, oreven an internal deletion, of the native molecule. A polynucleotidefragment of a dwf4 sequence will generally include at least about 15contiguous bases of the molecule in question, more preferably 18-25contiguous bases, even more preferably 30-50 or more contiguous bases ofthe dwf4 molecule, or any integer between 15 bases and the full-lengthsequence of the molecule. Fragments which provide at least one dwf4phenotype as defined above are useful in the production of transgenicplants. Fragments are also useful as oligonucleotide probes, to findadditional dwf4 sequences.

[0079] Similarly, a polypeptide fragment of a DWF4 molecule willgenerally include at least about 5-10 contiguous amino acid residues ofthe full-length molecule, preferably at least about 15-25 contiguousamino acid residues of the full-length molecule, and most preferably atleast about 20-50 or more contiguous amino acid residues of thefull-length DWF4 molecule, or any integer between 10 amino acids and thefull-length sequence of the molecule. Such fragments are useful for theproduction of antibodies and the like.

[0080] By “transgenic plant” is meant a plant into which one or moreexogenous polynucleotides have been introduced. Examples of means bywhich this can be accomplished are described below, and includeAgrobacterium-mediated transformation, biolistic methods,electroporation, and the like. In the context of the present invention,the transgenic plant contains a polynucleotide which is not normallypresent in the corresponding wild-type plant and which confers at leastone dwf4 phenotypic trait to the plant. The transgenic plant thereforeexhibits altered structure, morphology or biochemistry as compared witha progenitor plant which does not contain the transgene, when thetransgenic plant and the progenitor plant are cultivated under similaror equivalent growth conditions. Such a plant containing the exogenouspolynucleotide is referred to here as an R. generation transgenic plant.Transgenic plants may also arise from sexual cross or by selfing oftransgenic plants into which exogenous polynucleotides have beenintroduced. Such a plant containing the exogenous nucleic acid is alsoreferred to here as an R₁ generation transgenic plant. Transgenic plantswhich arise from a sexual cross with another parent line or by selfingare “descendants or the progeny” of a R₁ plant and are generally calledF_(n) plants or S_(n) plants, respectively, n meaning the number ofgenerations.

[0081] General Overview

[0082] In this report, we present morphological, biochemical, andmolecular analysis of a novel gene, dwf4, isolated from Arabidopsis.Morphologically, dwf4 plants display a dramatic reduction in the lengthof many different organs examined, and this size reduction isattributable to a defect in cell elongation. Biochemically, dwf4hypocotyls were converted completely to wild-type length with theapplication of BL, suggesting a deficiency in BRs. In agreement withthis, BR intermediate feeding analysis, indicated that dwf4 encodes acytochrome P450 that mediates multiple 22α-hydroxylation steps inbrassinosteriod biosynthesis. Sequencing of the dwf4 locus and analysisof the protein product are described.

[0083] The molecules of the present invention are therefore useful inthe production of transgenic plants which display at least one dwf4phenotype, so that the resulting plants have altered structure ormorphology. The present invention particularly provides for alteredstructure or morphology such as reduced cell length, extended floweringperiods, increased size of leaves or fruit, increased branching,increased seed production and altered sterol composition relativewild-type plants. The DWF4 polypeptides can be expressed to engineer aplant with desirable properties. The engineering is accomplished bytransforming plants with nucleic acid constructs described herein whichmay also comprise promoters and secretion signal peptides. Thetransformed plants or their progenies are screened for plants thatexpress the desired polypeptide.

[0084] Engineered plants exhibiting the desired altered structure ormorphology can be used in plant breeding or directly in agriculturalproduction or industrial applications. Plants having the alteredpolypeptide can be crossed with other altered plants engineered withalterations in other growth modulation enzymes, proteins or polypeptidesto produce lines with even further enhanced altered structuralmorphology characteristics compared to the parents or progenitor plants.

[0085] Isolation of Nucleic Acid Sequences from Plants

[0086] The isolation of dwf4 sequences from the polynucleotides of theinvention may be accomplished by a number of techniques. For instance,oligonucleotide probes based on the sequences disclosed here can be usedto identify the desired gene in a cDNA or genomic DNA library from adesired plant species. To construct genomic libraries, large segments ofgenomic DNA are generated by random fragmentation, e.g. usingrestriction endonucleases, and are ligated with vector DNA to formconcatemers that can be packaged into the appropriate vector. To preparea library of tissue-specific cDNAs, mRNA is isolated from tissues and acDNA library which contains the gene transcripts is prepared from themRNA.

[0087] The cDNA or genomic library can then be screened using a probebased upon the sequence of a cloned gene such as the polynucleotidesdisclosed here. Probes may be used to hybridize with genomic DNA or cDNAsequences to isolate homologous genes in the same or different plantspecies. Alternatively, the nucleic acids of interest can be amplifiedfrom nucleic acid samples using amplification techniques. For instance,polymerase chain reaction (PCR) technology to amplify the sequences ofthe genes directly from mRNA, from cDNA, from genomic libraries or cDNAlibraries. PCR.RTM. and other in vitro amplification methods may also beuseful, for example, to clone nucleic acid sequences that code forproteins to be expressed, to make nucleic acids to use as probes fordetecting the presence of the desired mRNA in samples, for nucleic acidsequencing, or for other purposes.

[0088] Appropriate primers and probes for identifying dwf4-specificgenes from plant tissues are generated from comparisons of the sequencesprovided herein. For a general overview of PCR see Innis et al. eds, PCRProtocols: A Guide to Methods and Applications, Academic Press, SanDiego (1990). Appropriate primers for this invention include, forinstance, those primers described in the Examples and Sequence Listings,as well as other primers derived from the dwf4 sequences disclosedherein. Suitable amplifications conditions may be readily determined byone of skill in the art in view of the teachings herein, for example,including reaction components and amplification conditions as follows:10 mM Tris-HCl, pH 8.3, 50 mM potassium chloride, 1.5 mM magnesiumchloride, 0.001% gelatin, 200 μM dATP, 200 μM dCTP, 200 μM dGTP, 200 μMdTTP, 0.4 μM primers, and 100 units per mL Taq polymerase; 96° C. for 3min., 30 cycles of 96° C. for 45 seconds, 50° C. for 60 seconds, 72° C.for 60 seconds, followed by 72° C. for 5 min.

[0089] Polynucleotides may also be synthesized by well-known techniquesas described in the technical literature. See, e.g., Carruthers, et al.(1982) Cold Spring Harbor Symp. Quant. Biol. 47:411-418, and Adams, etal. (1983) J. Am. Chem. Soc. 105:661. Double stranded DNA fragments maythen be obtained either by synthesizing the complementary strand andannealing the strands together under appropriate conditions, or byadding the complementary strand using DNA polymerase with an appropriateprimer sequence.

[0090] The polynucleotides of the present invention may also be used toisolate or create other mutant cell gene alleles. Mutagenesis consistsprimarily of site-directed mutagenesis followed by phenotypic testing ofthe altered gene product. Some of the more commonly employedsite-directed mutagenesis protocols take advantage of vectors that canprovide single stranded as well as double stranded DNA, as needed.Generally, the mutagenesis protocol with such vectors is as follows. Amutagenic primer, i.e., a primer complementary to the sequence to bechanged, but consisting of one or a small number of altered, added, ordeleted bases, is synthesized. The primer is extended in vitro by a DNApolymerase and, after some additional manipulations, the nowdouble-stranded DNA is transfected into bacterial cells. Next, by avariety of methods, the desired mutated DNA is identified, and thedesired protein is purified from clones containing the mutated sequence.For longer sequences, additional cloning steps are often requiredbecause long inserts (longer than 2 kilobases) are unstable in thosevectors. Protocols are known to one skilled in the art and kits forsite-directed mutagenesis are widely available from biotechnology supplycompanies, for example from Amersham Life Science, Inc. (ArlingtonHeights, Ill.) and Stratagene Cloning Systems (La Jolla, Calif.).

[0091] Control Elements

[0092] Regulatory regions can be isolated from the dwf4 gene and used inrecombinant constructs for modulating the expression of the dwf4 gene ora heterologous gene in vitro and/or in vivo. As shown in FIG. 10, thecoding region of the dwf4 gene (designated by the light grey bar) beginsat nucleotide position 1133. The region of the gene spanning nucleotidepositions 990-1132 of FIG. 10 includes the dwf4 promoter. This regionmay be used in its entirety or fragments of the region may be isolatedwhich provide the ability to direct expression of a coding sequencelinked thereto.

[0093] Thus, promoters can be identified by analyzing the 5′ sequencesof a genomic clone corresponding to the dwf4-specific genes describedhere. Sequences characteristic of promoter sequences can be used toidentify the promoter. Sequences controlling eukaryotic gene expressionhave been extensively studied. For instance, promoter sequence elementsinclude the TATA box consensus sequence (TATAAT), which is usually 20 to30 base pairs upstream of the transcription start site. In mostinstances the TATA box is required for accurate transcriptioninitiation. In plants, further upstream from the TATA box, at positions−80 to −100, there is typically a promoter element with a series ofadenines surrounding the trinucleotide G (or T) N G. (See, J. Messing etal., in Genetic Engineering in Plants, pp. 221-227 (Kosage, Meredith andHollaender, eds. (1983)). Methods for identifying and characterizingpromoter regions in plant genomic DNA are described, for example, inJordano et al. (1989) Plant Cell 1:855-866; Bustos et al (1989) PlantCell 1:839-854; Green et al. (1988) EMBO J. 7:4035-4044; Meier et al.(1991) Plant Cell 3:309-316; and Zhang et al (1996) Plant Physiology110:1069-1079).

[0094] Additionally, the promoter region may include nucleotidesubstitutions, insertions or deletions that do not substantially affectthe binding of relevant DNA binding proteins and hence the promoterfunction. It may, at times, be desirable to decrease the binding ofrelevant DNA binding proteins to “silence” or “down-regulate” apromoter, or conversely to increase the binding of relevant DNA bindingproteins to “enhance” or “up-regulate” a promoter. In such instances,the nucleotide sequence of the promoter region may be modified by, e.g.,inserting additional nucleotides, changing the identity of relevantnucleotides, including use of chemically-modified bases, or by deletingone or more nucleotides.

[0095] Promoter function can be assayed by methods known in the art,preferably by measuring activity of a reporter gene operatively linkedto the sequence being tested for promoter function. Examples of reportergenes include those encoding luciferase, green fluorescent protein, GUS,neo, cat and bar.

[0096] Polynucleotides comprising untranslated (UTR) sequences andintron/exon junctions are also within the scope of the invention. UTRsequences include introns and 5′ or 3′ untranslated regions (5′ UTRs or3′ UTRs). As shown in FIGS. 2 and 10, the dwf4 gene sequence includeseight exons and seven introns. These portions of the dwf4 geneespecially UTRs, can have regulatory functions related to, for example,translation rate and mRNA stability. Thus, these portions of the genecan be isolated for use as elements of gene constructs for expression ofpolynucleotides encoding desired polypeptides. The 5′ control elementregion of dwf4 extends from nucleotides 1 through 3202 of SEQ ID NO:1.Further, as described in Example 11, a 1.1 kb portion of this regionthat is directly upstream of the translation initiation site containselements necessary for transcriptional control of dwf4. In contrast, a280 bp fragment of the dwf4 control element region that includes theTATA-like region does nQt appear to contain all of the necessarytranscriptional control elements (see, Example 11).

[0097] Introns of genomic DNA segments may also have regulatoryfunctions. Sometimes promoter elements, especially transcriptionenhancer or suppressor elements, are found within introns. Also,elements related to stability of heteronuclear RNA and efficiency oftransport to the cytoplasm for translation can be found in intronelements. Thus, these segments can also find use as elements ofexpression vectors intended for use to transform plants.

[0098] The introns, UTR sequences and intron/exon junctions can varyfrom the native sequence. Such changes from those sequences preferablywill not affect the regulatory activity of the UTRs or intron orintron/exon junction sequences on expression, transcription, ortranslation. However, in some instances, down-regulation of suchactivity may be desired to modulate traits or phenotypic or in vitroactivity.

[0099] Use of Nucleic Acids of the Invention to Inhibit Gene Expression

[0100] The isolated sequences prepared as described herein, can be usedto prepare expression cassettes useful in a number of techniques. Forexample, expression cassettes of the invention can be used to suppress(underexpress) endogenous dwf4 gene expression. Inhibiting expressioncan be useful, for instance, in suppressing the phenotype (e.g., dwarfappearance, 22α-hydroxylase activity) exhibited by dwf4 plants. Further,the inhibitory polynucleotides of the present invention can also be usedin combination with overexpressing constructs described below, forexample, using suitable tissue-specific promoters linked topolynucleotides described herein. In this way, the polynucleotides canbe used to promote dwf4 phenotypes (e.g., activity) in selected tissueand, at the same time, inhibit dwf4 phenotypes (e.g., activity) indifferent tissue(s).

[0101] A number of methods can be used to inhibit gene expression inplants. For instance, antisense technology can be conveniently used. Toaccomplish this, a nucleic acid segment from the desired gene is clonedand operably linked to a promoter such that the antisense strand of RNAwill be transcribed. The expression cassette is then transformed intoplants and the antisense strand of RNA is produced. In plant cells, ithas been suggested that antisense RNA inhibits gene expression bypreventing the accumulation of mRNA which encodes the enzyme ofinterest, see, e.g., Sheehy et al (1988) Proc. Nat. Acad. Sci. USA85:8805-8809, and Hiatt et al., U.S. Pat. No. 4,801,340.

[0102] The nucleic acid segment to be introduced generally will besubstantially identical to at least a portion of the endogenous gene orgenes to be repressed. The sequence, however, need not be perfectlyidentical to inhibit expression. The vectors of the present inventioncan be designed such that the inhibitory effect applies to otherproteins within a family of genes exhibiting homology or substantialhomology to the target gene.

[0103] For antisense suppression, the introduced sequence also need notbe full length relative to either the primary transcription product orfully processed mRNA. Generally, higher homology can be used tocompensate for the use of a shorter sequence. Furthermore, theintroduced sequence need not have the same intron or exon pattern, andhomology of non-coding segments may be equally effective. Normally, asequence of between about 30 or 40 nucleotides and about full lengthnucleotides should be used, though a sequence of at least about 100nucleotides is preferred, a sequence of at least about 200 nucleotidesis more preferred, and a sequence of at least about 500 nucleotides isespecially preferred. It is to be understood that any integer betweenthe above-recited ranges is intended to be captured herein.

[0104] Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of dwf4 genes. It is possible to design ribozymes thatspecifically pair with virtually any target RNA and cleave thephosphodiester backbone at a specific location, thereby functionallyinactivating the target RNA. In carrying out this cleavage, the ribozymeis not itself altered, and is thus capable of recycling and cleavingother molecules, making it a true enzyme. The inclusion of ribozymesequences within antisense RNAs confers RNA-cleaving activity upon them,thereby increasing the activity of the constructs.

[0105] A number of classes of ribozymes have been identified. One classof ribozymes is derived from a number of small circular RNAs which arecapable of self-cleavage and replication in plants. The RNAs replicateeither alone (viroid RNAs) or with a helper virus (satellite RNAs).Examples include RNAs from avocado sunblotch viroid and the satelliteRNAs from tobacco ringspot virus, lucerne transient streak virus, velvettobacco mottle virus, solanum nodiflorum mottle virus and subterraneanclover mottle virus. The design and use of target RNA-specific ribozymesis described in Haseloff et al (1988) Nature 334:585-591.

[0106] Another method of suppression is sense suppression. Introductionof expression cassettes in which a nucleic acid is configured in thesense orientation with respect to the promoter has been shown to be aneffective means by which to block the transcription of target genes. Foran example of the use of this method to 15- modulate expression ofendogenous genes see, Napoli et al (1990) The Plant Cell 2:279-289 andU.S. Pat. Nos. 5,034,323, 5,231,020, and 5,283,184.

[0107] Generally, where inhibition of expression is desired, sometranscription of the introduced sequence occurs. The effect may occurwhere the introduced sequence contains no coding sequence per se, butonly intron or untranslated sequences homologous to sequences present inthe primary transcript of the endogenous sequence. The introducedsequence generally will be substantially identical to the endogenoussequence intended to be repressed. This minimal identity will typicallybe greater than about 50%-65%, but a higher identity might exert a moreeffective repression of expression of the endogenous sequences.Substantially greater identity of more than about 80% is preferred,though about 95% to absolute identity would be most preferred. It is tobe understood that any integer between the above-recited ranges isintended to be captured herein. As with antisense regulation, the effectshould apply to any other proteins within a similar family of genesexhibiting homology or substantial homology.

[0108] For sense suppression, the introduced sequence in the expressioncassette, needing less than absolute identity, also need not be fulllength, relative to either the primary transcription product or fullyprocessed mRNA. This may be preferred to avoid concurrent production ofsome plants which are overexpressers. A higher identity in a shorterthan full length sequence compensates for a longer, less identicalsequence. Furthermore, the introduced sequence need not have the sameintron or exon pattern, and identity of non-coding segments will beequally effective. Normally, a sequence of the size ranges noted abovefor antisense regulation is used.

[0109] Use of Nucleic Acids of the Invention to Enhance Gene Expression

[0110] In addition to inhibiting certain features of a plant, thepolynucleotides of the invention can be used to increase certainfeatures such as extending flowering, producing larger leaves or fruit,producing increased branching and increasing seed production. This canbe accomplished by the overexpression of dwf4 polynucleotides.

[0111] The exogenous dwf4 polynucleotides do not have to code for exactcopies of the endogenous dwf4 proteins. Modified DWF4 protein chains canalso be readily designed utilizing various recombinant DNA techniqueswell known to those skilled in the art and described for instance, inSambrook et al., supra. Hydroxylamine can also be used to introducesingle base mutations into the coding region of the gene (Sikorski et al(1991) Meth. Enzymol. 194: 302-318). For example, the chains can varyfrom the naturally occurring sequence at the primary structure level byamino acid substitutions, additions, deletions, and the like. Thesemodifications can be used in a number of combinations to produce thefinal modified protein chain.

[0112] It will be apparent that the polynucleotides described herein canbe used in a variety of combinations. For example, the polynucleotidescan be used to produce different phenotypes in the same organism, forinstance by using tissue-specific promoters to overexpress a dwf4polynucleotide in certain tissues (e.g., leaf tissue) while at the sametime using tissue-specific promoters to inhibit expression of dwf4 inother tissues. In addition, fusion proteins of the polynucleotidesdescribed herein with other known polynucleotides (e.g., polynucleotidesencoding products involved in the BR pathway) can be constructed andemployed to obtain desired phenotypes.

[0113] Any of the dwf4 polynucleotides described herein can also be usedin standard diagnostic assays, for example, in assays mRNA levels (see,Sambrook et al, supra); as hybridization probes, e.g., in combinationwith appropriate means, such as a label, for detecting hybridization(see, Sambrook et al., supra); as primers, e.g., for PCR (see, Sambrooket al., supra); attached to solid phase supports and the like.

[0114] Preparation of Recombinant Vectors

[0115] To use isolated sequences in the above techniques, recombinantDNA vectors suitable for transformation of plant cells are prepared.Techniques for transforming a wide variety of higher plant species arewell known and described further below as well as in the technical andscientific literature. See, for example, Weising et al (1988) Ann. Rev.Genet. 22:421-477. A DNA sequence coding for the desired polypeptide,for example a cDNA sequence encoding the full length DWF4 protein, willpreferably be combined with transcriptional and translational initiationregulatory sequences which will direct the transcription of the sequencefrom the gene in the intended tissues of the transgenic plant.

[0116] Such regulatory elements include but are not limited to thepromoters derived from the genome of plant cells (e.g., heat shockpromoters such as soybean hsp17.5-E or hsp17.3-B (Gurley et al. (1986)Mol. Cell. Biol. 6:559-565); the promoter for the small subunit ofRUBISCO (Coruzzi et al. (1984) EMBO J. 3:1671-1680; Broglie et al (1984)Science 224:838-843); the promoter for the chlorophyll a/b bindingprotein) or from plant viruses viral promoters such as the ³⁵S RNA and19S RNA promoters of CaMV (Brisson et al. (1984) Nature 310:511-514), orthe coat protein promoter of TMV (Takamatsu et al. (1987) EMBO J.6:307-311), cytomegalovirus hCMV immediate early gene, the early or latepromoters of SV40 adenovirus, the lac system, the trp system, the TACsystem, the TRC system, the major operator and promoter regions of phageA, the control regions of fd coat protein, the promoter for3-phosphoglycerate kinase, the promoters of acid phosphatase, heat shockpromoters (e.g., as described above) and the promoters of the yeastalpha-mating factors.

[0117] In construction of recombinant expression cassettes of theinvention, a plant promoter fragment may be employed which will directexpression of the gene in all tissues of a regenerated plant. Suchpromoters are referred to herein as “constitutive” promoters and areactive under most environmental conditions and states of development orcell differentiation. Examples of constitutive promoters include thecauliflower mosaic virus (CaMV) ³⁵S transcription initiation region, theT-DNA mannopine synthetase promoter (e.g., the 1′- or 2′-promoterderived from T-DNA of Agrobacterium tumafaciens), and othertranscription initiation regions from various plant genes known to thoseof skill.

[0118] Alternatively, the plant promoter may direct expression of thepolynucleotide of the invention in a specific tissue (tissue-specificpromoters) or may be otherwise under more precise environmental control(inducible promoters). Examples of tissue-specific promoters underdevelopmental control include promoters that initiate transcription onlyin certain tissues, such as fruit, seeds, or flowers such as tissue- ordevelopmental-specific promoter, such as, but not limited to the dwf4promoter, the CHS promoter, the PATATIN promoter, etc. The tissuespecific E8 promoter from tomato is particularly useful for directinggene expression so that a desired gene product is located in fruits.

[0119] Other suitable promoters include those from genes encodingembryonic storage proteins. Examples of environmental conditions thatmay affect transcription by inducible promoters include anaerobicconditions, elevated temperature, or the presence of light. If properpolypeptide expression is desired, a polyadenylation region at the3′-end of the coding region should be included. The polyadenylationregion can be derived from the natural gene, from a variety of otherplant genes, or from T-DNA. In addition, the promoter itself can bederived from the dwf4 gene, as described above.

[0120] The vector comprising the sequences (e.g., promoters or codingregions) from genes of the invention will typically comprise a markergene which confers a selectable phenotype on plant cells. For example,the marker may encode biocide resistance, particularly antibioticresistance, such as resistance to kanamycin, G418, bleomycin,hygromycin, or herbicide resistance, such as resistance tochlorosluforon or Basta.

[0121] Production of Transgenic Plants

[0122] DNA constructs of the invention may be introduced into the genomeof the desired plant host by a variety of conventional techniques. Forreviews of such techniques see, for example, Weissbach & WeissbachMethods for Plant Molecular Biology (1988, Academic Press, N.Y.) SectionVIII, pp. 421-463; and Grierson & Corey, Plant Molecular Biology (1988,2d Ed.), Blackie, London, Ch. 7-9. For example, the DNA construct may beintroduced directly into the genomic DNA of the plant cell usingtechniques such as electroporation and microinjection of plant cellprotoplasts, or the DNA constructs can be introduced directly to planttissue using biolistic methods, such as DNA particle bombardment (see,e.g., Klein et al (1987) Nature 327:70-73). Alternatively, the DNAconstructs may be combined with suitable T-DNA flanking regions andintroduced into a conventional Agrobacterium tumefaciens host vector.Agrobacterium tumefaciens-mediated transformation techniques, includingdisarming and use of binary vectors, are well described in thescientific literature. See, for example Horsch et al (1984) Science233:496-498, and Fraley et al (1983) Proc. Nat'l. Acad. Sci. USA80:4803. The virulence functions of the Agrobacterium tumefaciens hostwill direct the insertion of the construct and adjacent marker into theplant cell DNA when the cell is infected by the bacteria using binary TDNA vector (Bevan (1984) Nuc. Acid Res. 12:8711-8721) or theco-cultivation procedure (Horsch et al (1985) Science 227:1229-1231).Generally, the Agrobacterium transformation system is used to engineerdicotyledonous plants (Bevan et al (1982) Ann. Rev. Genet 16:357-384;Rogers et al (1986) Methods Enzymol. 118:627-641). The Agrobacteriumtransformation system may also be used to transform, as well astransfer, DNA to monocotyledonous plants and plant cells. (seeHernalsteen et al (1984) EMBO J3:3039-3041; Hooykass-Van Slogteren et al(1984) Nature 311:763-764; Grimsley et al (1987) Nature 325:1677-179;Boulton et al (1989) Plant Mol. Biol. 12:31-40.; and Gould et al (1991)Plant Physiol. 95:426-434).

[0123] Alternative gene transfer and transformation methods include, butare not limited to, protoplast transformation through calcium-,polyethylene glycol (PEG)- or electroporation-mediated uptake of nakedDNA (see Paszkowski et al. (1984) EMBO J3:2717-2722, Potrykus et al.(1985) Molec. Gen. Genet. 199:169-177; Fromm et al. (1985) Proc. Nat.Acad. Sci. USA 82:5824-5828; and ShimamotQ (1989) Nature 338:274-276)and electroporation of plant tissues (D'Halluin et al. (1992) Plant Cell4:1495-1505). Additional methods for plant cell transformation includemicroinjection, silicon carbide mediated DNA uptake (Kaeppler et al.(1990) Plant Cell Reporter 9:415-418), and microprojectile bombardment(see Klein et al. (1988) Proc. Nat. Acad. Sci. USA 85:4305-4309; andGordon-Kamm et al. (1990) Plant Cell 2:603-618).

[0124] Transformed plant cells which are produced by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype and thus the desired phenotype.Such regeneration techniques rely on manipulation of certainphytohormones in a tissue culture growth medium, typically relying on abiocide and/or herbicide marker which has been introduced together withthe desired nucleotide sequences. Plant regeneration from culturedprotoplasts is described in Evans, et al., “Protoplasts Isolation andCulture” in Handbook of Plant Cell Culture, pp. 124-176, MacmillianPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, pollens,embryos or parts thereof. Such regeneration techniques are describedgenerally in Klee et al (1987) Ann. Rev. of Plant Phys. 38:467-486.

[0125] The nucleic acids of the invention can be used to confer desiredtraits on essentially any plant. A wide variety of plants and plant cellsystems may be engineered for the desired physiological and agronomiccharacteristics described herein using the nucleic acid constructs ofthe present invention and the various transformation methods mentionedabove. In preferred embodiments, target plants and plant cells forengineering include, but are not limited to, those monocotyledonous anddicotyledonous plants, such as crops including grain crops (e.g., wheat,maize, rice, millet, barley), fruit crops (e.g., tomato, apple, pear,strawberry, orange), forage crops (e.g., alfalfa), root vegetable crops(e.g., carrot, potato, sugar beets, yam), leafy vegetable crops (e.g.,lettuce, spinach); flowering plants (e.g., petunia, rose,chrysanthemum), conifers and pine trees (e.g., pine fir, spruce); plantsused in phytoremediation (e.g., heavy metal accumulating plants); oilcrops (e.g., sunflower, rape seed) and plants used for experimentalpurposes (e.g., Arabidopsis). Thus, the invention has use over a broadrange of plants, including, but not limited to, species from the generaAsparagus, Avena, Brassica, Citrus, Citrullus, Capsicum, Cucurbita,Daucus, Glycine, Hordeum, Lactuca, Lycopersicon, Malus, Manihot,Nicotiana, Oryza, Persea, Pisum, Pyrus, Prunus, Raphanus, Secale,Solanum, Sorghum, Triticum, Vitis, Vigna, and Zea.

[0126] One of skill in the art will recognize that after the expressioncassette is stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed.

[0127] A transformed plant cell, callus, tissue or plant may beidentified and isolated by selecting or screening the engineered plantmaterial for traits encoded by the marker genes present on thetransforming DNA. For instance, selection may be performed by growingthe engineered plant material on media containing an inhibitory amountof the antibiotic or herbicide to which the transforming gene constructconfers resistance. Further, transformed plants and plant cells may alsobe identified by screening for the activities of any visible markergenes (e.g., the β-glucuronidase, luciferase, B or C1 genes) that may bepresent on the recombinant nucleic acid constructs of the presentinvention. Such selection and screening methodologies are well known tothose skilled in the art.

[0128] Physical and biochemical methods also may be used to identifyplant or plant cell transformants containing the gene constructs of thepresent invention. These methods include but are not limited to: 1)Southern analysis or PCR amplification for detecting and determining thestructure of the recombinant DNA insert; 2) Northern blot, S1 RNaseprotection, primer-extension or reverse transcriptase-PCR amplificationfor detecting and examining RNA transcripts of the gene constructs; 3)enzymatic assays for detecting enzyme or ribozyme activity, where suchgene products are encoded by the gene construct; 4) protein gelelectrophoresis, Western blot techniques, immunoprecipitation, orenzyme-linked immunoassays, where the gene construct products areproteins. Additional techniques, such as in situ hybridization, enzymestaining, and immunostaining, also may be used to detect the presence orexpression of the recombinant construct in specific plant organs andtissues. The methods for doing all these assays are well known to thoseskilled in the art.

[0129] Effects of gene manipulation using the methods of this inventioncan be observed by, for example, northern blots of the RNA (e.g., mRNA)isolated from the tissues of interest. Typically, if the amount of mRNAhas increased, it can be assumed that the endogenous dwf4 gene is beingexpressed at a greater rate than before. Other methods of measuring DWF4activity can be used. For example, cell length can be measured atspecific times. Because dwf4 affects the BR biosynthetic pathway, anassay that measures the amount of BL can also be used. Such assays areknown in the art. Different types of enzymatic assays can be used,depending on the substrate used and the method of detecting the increaseor decrease of a reaction product or by-product. In addition, the levelsof DWF4 protein expressed can be measured immunochemically, i.e., ELISA,RIA, EIA and other antibody based assays well known to those of skill inthe art, by electrophoretic detection assays (either with staining orwestern blotting), and sterol (BL) detection assays.

[0130] The transgene may be selectively expressed in some tissues of theplant or at some developmental stages, or the transgene may be expressedin substantially all plant tissues, substantially along its entire lifecycle. However, any combinatorial expression mode is also applicable.

[0131] The present invention also encompasses seeds of the transgenicplants described above wherein the seed has the transgene or geneconstruct. The present invention further encompasses the progeny,clones, cell lines or cells of the transgenic plants described abovewherein said progeny, clone, cell line or cell has the transgene or geneconstruct.

[0132] Polypeptides

[0133] The present invention also includes DWF4 polypeptides, includingsuch polypeptides as a fusion, or chimeric protein product (comprisingthe protein, fragment, analogue, mutant or derivative joined via apeptide bond to a heterologous protein sequence (of a differentprotein)). Such a chimeric product can be made by ligating theappropriate nucleic acid sequences encoding the desired amino acidsequences to each other by methods known in the art, in the propercoding frame, and expressing the chimeric product by methods commonlyknown in the art.

[0134] As noted above, DWF4 phenotype includes any macroscopic,microscopic or biochemical changes which are characteristic of over- orunder-expression of dwf4. Thus, DWF4 polypeptide phenotype (e.g.,activities) can include any activity that is exhibited by the nativeDWF4 polypeptide including, for example, in vitro, in vivo, biological,enzymatic, immunological, substrate binding activities, etc.Non-limiting examples of DWF4 activities include:

[0135] (a) activities displayed by other heme-thiolate enzymes;

[0136] (b) characteristic Soret absorption peak at 40 nm when thesubstrate-bound reduced form is exposed to the lights (see, e.g.,Jefcoate et al., infra);

[0137] (c) hydroxylation of various substrates via monooxygenaseactivity, which utilizes molecular oxygen and reducing equivalents fromNAD(P)H;

[0138] (d) oxidation, dealkylation, deaminoation, dehalogenation, andsulfoxide formation that are involved in a variety of biological eventsin plants and animals (e.g., catabolism, anabolism, and xenobioticactivities);

[0139] (e) recognition of at two substrates: campestanol (CN) and6-deoxocastasterone (6-deoxoCS);

[0140] (f) 22α-hydroxylase activity;

[0141] (g) DWF4 phenotypic activities such as modulation of cell length,periods of flowering, branching, seed production, leaf size, and sterolcomposition in a plant;

[0142] (h) regulation of gibberellic acid, cytokinins and/or auxin;

[0143] (i) induce resistance to plant pathogens (see, e.g., U.S. Pat.No. 5,952,545);

[0144] (j) accelerating growth at low temperatures; and

[0145] (k) accelerating growth in dark conditions.

[0146] A DWF4 analog, whether a derivative, fragment or fusion of nativeDWF4 polypeptides, is capable of at least one DWF4 activity. Preferably,the analogs exhibit at least 60% of the activity of the native protein,more preferably at least 70% and even more preferably at least 80%, 85%,90% or 95% of at least one activity of the native protein.

[0147] Further, such analogs exhibit some sequence identity to thenative DWF4 polypeptide sequence. Preferably, the variants will exhibitat least 35%, more preferably at least 59%, even more preferably 75% or80% sequence identity, even more preferably 85% sequence identity, evenmore preferably, at least 90% sequence identity; more preferably atleast 95%, 96%, 97%, 98% or 99% sequence identity.

[0148] DWF4 analogs can include derivatives with increased or decreasedactivities as compared to the native DWF4 polypeptides. Such derivativescan include changes within the domains, motifs and/or consensus regionsof the native DWF4 polypeptide, which are described in detail in Example3.

[0149] Once class of analogs is those polypeptide sequences that differfrom the native DWF4 polypeptide by changes, insertions, deletions, orsubstitution; at positions flanking the domain and/or conservedresidues. For example, an analog can comprise (1) the domains of a DWF4polypeptide and/or (2) residues conserved between the DWF4 polypeptideand other cytochrome P450 proteins, for example as shown in FIG. 3 anddescribed in Example 3.

[0150] Another class of analogs includes those that comprise a DWF4polypeptide sequence that differs from the native sequence in the domainof interest or conserved residues by a conservative substitution. Forexample, an analog that exhibits increased sterol binding can haveoptimized sterol binding domain sequences that differ from the nativesequence.

[0151] Yet another class of analogs includes those that lack one of thein vitro activities or structural features of the native DWF4polypeptides, for example, dominant negative mutants or analogs thatcomprise a heme-binding domain but contain an inactivated steroidbinding domain.

[0152] DWF4 polypeptide fragments can comprise sequences from the nativeor analog sequences, for example fragments comprising one or more of thefollowing P450 domains or regions: A, B, C, D, anchor binding, andproline rich. Such domains and regions are shown in FIGS. 2B, 3 anddescribed in Example 3.

[0153] Fusion polypeptides comprising DWF4 polypeptides (e.g., native,analogs, or fragments thereof) can also be constructed. Non-limitingexamples of other polypeptides that can be used in fusion proteinsinclude chimeras of DWF4 polypeptides and fragments thereof; and P450polypeptides or fragments thereof, such as those shown in FIG. 3.

[0154] In addition, DWF4 polypeptides, derivatives (including fragmentsand chimeric proteins), mutants and analogues can be chemicallysynthesized. See, e.g., Clark-Lewis et al. (1991) Biochem. 30:3128-3135and Merrifield (1963) J. Amer. Chem. Soc. 85:2149-2156. For example,DWF4, derivatives, mutants and analogues can be synthesized by solidphase techniques, cleaved from the resin, and purified by preparativehigh performance liquid chromatography (e.g., see Creighton, 1983,Proteins, Structures and Molecular Principles, W. H. Freeman and Co.,N.Y., pp. 50-60). DWF4, derivatives and analogues that are proteins canalso be synthesized by use of a peptide synthesizer. The composition ofthe synthetic peptides may be confirmed by amino acid analysis orsequencing (e.g., the Edman degradation procedure; see Creighton, 1983,Proteins, Structures and Molecular Principles, W. H. Freeman and Co.,N.Y., pp. 34-49).

[0155] Further, the dwf4 polynucleotides and DWF4 polypeptides describedherein can be used to generate antibodies that specifically recognizeand bind to the protein products of the dwf4 polynucleotides. (See,Harlow and Lane, eds. (1988) “Antibodies: A Laboratory Manual”). TheDWF4 polypeptides and antibodies thereto can also be used in standarddiagnostic assays, for example, radioimmunoassays, ELISA (enzyme linkedimmunoradiometric assays), “sandwich” immunoassays, immunoradiometricassays, in situ immunoassay, western blot analysis, immunoprecipitationassays, immunofluorescent assays and PAGE-SDS.

[0156] Applications

[0157] The present invention finds use in various applications, forexample, including but not limited to those listed above.

[0158] The polynucleotide sequences may additionally be used to isolatemutant dwf4 gene alleles. Such mutant alleles may be isolated from plantspecies either known or proposed to have a genotype which contributes toaltered plant morphology. Additionally, such plant dwf4 gene sequencescan be used to detect plant dwf4 gene regulatory (e.g., promoter orpromoter/enhancer) defects which can affect plant growth.

[0159] The molecules of the present invention can be used to provideplants with increased seed and/fruit production, extended floweringperiods and increased branching. The molecules described herein can beused to alter the sterol composition of a plant, thereby increasing orreducing cholesterol content in the plant. A still further utility ofthe molecules of the present invention is to provide a tool for studyingthe biosynthesis of brassinosteriods, both in vitro and in vivo.

[0160] The dwf4 gene of the invention also has utility as a transgeneencoding a cytochrome P450 protein that mediates multiple 22αhydroxylation steps in brassinosteriod biosynthesis which results in atransgenic plant to alter plant structure or morphology. The dwf4 genealso has utility for encoding the DWF4 protein in recombinant vectorswhich may be inserted into host cells to express the DWF4 protein.Further, the dwf4 polynucleotides of the invention may be utilized (1)as nucleic acid probes to screen nucleic acid libraries to identifyother enzymatic genes or mutants; (2) as nucleic acid sequences to bemutated or modified to produce DWF4 protein variants or derivatives; (3)as nucleic acids encoding 22α-hydroxylase in molecular biologytechniques or industrial applications commonly known to those skilled inthe art.

[0161] The dwf4 nucleic acid molecules may be used to design plant dwf4antisense molecules, useful, for example, in plant dwf4 gene regulationor as antisense primers in amplification reactions of plant dwf4 genenucleic acid sequences. With respect to plant dwf4 gene regulation, suchtechniques can be used to regulate, for example, plant growth,development or gene expression. Further, such sequences may be used aspart of ribozyme and/or triple helix sequences, also useful for dwf4gene regulation.

[0162] The dwf4 control element (e.g., promoter) of the presentinvention may be utilized as a plant promoter to express any protein,polypeptide or peptide of interest in a transgenic plant. In particular,the dwf4 promoter may be used to express a protein involved inbrassinosteriod biosynthesis.

[0163] The Arabidopsis DWF4 protein of the invention can be used in anybiochemical applications (experimental or industrial) where22α-hydroxylase activity is desired, for example, but not limited to,regulation of BL synthesis, regulation of other sterol synthesis,modification of elongating plant structures, and experimental orindustrial biochemical applications known to those skilled in the art.

EXPERIMENTAL

[0164] Below are examples of specific embodiments for carrying out thepresent invention. The examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way.

[0165] Efforts have been made to ensure accuracy with respect to numbersused (e.g., amounts, temperatures, etc.), but some experimental errorand deviation should, of course, be allowed for.

Example 1 Materials and Methods

[0166] A. Plant Growth Conditions

[0167] The conditions used for plant growth were essentially asdescribed preyiously (Feldmann (1991) Plant J 1:71-82; Forsthoefel etal. (1992) Aust. J. Plant Physiol. 19:353-366), except thatagar-solidified medium contained 0.5% sucrose. Seedlings up to 2 weeksof age (6 weeks of age for dark-growth experiments) were grown on 0.8%agar-solidified medium containing 1× Murashige and Skoog 1962 salts(Murashige, T., and Skoog, F. (1962) Physiol. Plant. 15:473-497) and0.5% sucrose (w/v) and cold treated (4° C.) for 2 days in the darkbefore transfer to the light (24 hr light; 80 μmol m⁻² sec⁻¹); olderplants were grown in potting soil. The plates were sealed with Parafilm(American National Can Co., Chicago, Ill.) for the entire experiment.For nucleic acid extraction, genetic analysis, and other experiments inwhich mature plants were required, seeds were sown on Metromix 350(Grace Sierra, Milpitas, Calif.) presoaked with distilled water. Thepots were covered with plastic wrap and cold treated (4° C.) for twodays before transfer to a growth chamber (16:8, light [240 μmol m⁻²sec⁻¹]:dark; 22 and 21° C., respectively, and 75 to 90% humidity). Theplastic wrap was removed 5 days after germination, and the pots weresubirrigated in distilled water as required. Germination of seeds fordark growth experiments was induced by overnight exposure of the seedsto light immediately after removing the plates from incubation at 4° C.The dwf4-1 and dwf4-2 mutations were in the Arabidopsis thaliana ecotypeWassilewskija (Ws-2) background; the dwf4-3 and dwf4-4 mutations were inthe Enkheim (En-2) background.

[0168] B. Analytical Methods

[0169] Protoplasts were obtained by overnight incubation of slicedleaves in 0.1% cellulysin, 0.1% driselase, 0.1% macerase (Calbiochem,San Diego, Calif.) in 125 mM Mes, pH 5.8, 0.5 M mannitol, and 7 mM CaCl₂(Galbraith et al. (1992) Planta 186:324-326. Immediately beforeobservation, chloroplasts were stained with a solution of 1.5% KI and 1%12. Measurements were performed as described for tissue sections, andplane areas were calculated according to the formula A=πr².

[0170] Chlorophyll determinations were performed from 2-week-oldsoil-grown plants. Green tissue was weighed, frozen in liquid nitrogen,and extracted in dim light with 80% acetone in the presence of a mixtureof equal parts sand, NaHCO₃, and Na₂SO₄. After brief centrifugation, thesupernatant was collected and the extraction was repeated twice, poolingthe supernatants from each sample. Chlorophylls a and b were measuredspectrophotometrically, as described in Chory et al. (1991), supra.

[0171] C. Growth Signal Response Measurements

[0172] Gibberellic acid (GA) response was assayed on plants grownindividually in 5.7-cm pots. Once inflorescences reached 1 to 2 mm inheight, they were sprayed weekly with 1 mM GA3 (Sigma). Control plantswere sprayed with water. One week after the third spraying, plants werecollected, and the length of the main stem was measured between the topof the rosette and the base of the most distal pedicel; 13 to 18 plantsof each line were measured per treatment. Auxin response was tested bygrowing seedlings for 10 days under 16 hr of light on verticallyoriented agar plates containing various concentrations of 2,4-D (Gibco,Grand Island, N.Y.). Genetic interaction with the hy2 mutation wastested by growing seedlings under continuous light for 7 days.Brassinolide (BL) response was determined in liquid culture, asdescribed by Clouse et al. (1993), supra, except that three or fourseedlings were grown in each well of a 24-well culture plate for 7 days.Measurements were taken for 10 to 20 seedlings for each genotype andcondition, under a dissection microscope fitted with an ocularmicrometer.

[0173] D. Microscopy

[0174] Tissues were fixed in 2% glutaraldehyde and 0.05 M sodiumcacodylate, pH 6.9, for 2 hr at room temperature or overnight at 4° C.,followed by three washes in buffer. For light microscopy, 1% safraninwas included in the first wash, and embedding was performed in ParaplastPlus (Oxford Labware, St. Louis, Mo.). Ten-millimeter sections from fiveindividual plants per line were analyzed and photographed, and cellmeasurements were taken using a ruler on 5×7 inch prints. A print of ahemocytometer grid at the same final magnification was used forcalibration. At least 25 cells were measured per sample, with a minimumof 150 cells per line. For electron microscopy, the tissues were treatedafter fixation with 1% tannic acid in buffer for 30 min, washed threetimes, and postfixed in 1% OsO₄ in buffer for 2 hr, followed by fivewashes and dehydration through an ethanol series. Samples fortransmission electron microscopy were embedded in Spurr's resin.Sections (90 nm) were stained with saturated uranyl acetate followed byReynolds's lead citrate (Reynolds (1963) J. Cell Biol. 17:208-212) andexamined in a JEOL (Tokyo, Japan) 100-CX instrument. For scanningelectron microscopy, samples were transferred to freon 113, criticalpoint dried, and sputter-coated with 30 to 50 nm of gold. Analysis wasperformed in a microscope (ISI model DS 130; Topcon, Inc., Paramus,N.J.) with an accelerating voltage of 15 kV. Electron microscopy wasperformed at the Electron Microscope Facility, Division ofBiotechnology, Arizona Research Laboratories, University of Arizona.

Example 2 Isolation of dwf4 Gene

[0175] A. Isolation of the DWF4 Gene

[0176] The dwf4-1 mutation was identified in a screen of 14,000transformants of Arabidopsis, resulting in a dwarfed phenotype similarto dwf1 (Feldmann and Marks (1987) Mol. Gen. Genet. 208:1-9; Feldmann etal. (1989) Science 243:1351-1354; referred to as diminuto in Takahashiet al. (1995) Genes Dev. 9:97-107 and Szekeres et al., supra) and det2(Azpiroz et al. (1998), supra). Two independent lines were found thatsegregated for a similar phenotype: both were shorter than dwfl, buttheir rosette diameter was comparable to that mutant. These dwarfs werealso essentially infertile. The most striking aspect of the morphologyof these mutants is their similarity to det2 (Chory et al. (1991) PlantCell 3:445-459). For this reason, further analysis was conducted withthese lines. After being found to be allelic to each other, both weredesignated as dwf4.

[0177] dwf4-1 segregated for a single kanamycin resistance marker, andgel blot analysis with DNA from single plants of this family confirmedthat the pattern is consistent with a single insert. The dwf4 mutationwas subsequently shown to be inherited as a monogenic, recessiveMendelian trait that, in dwf4-1, cosegregates with the dominantkanamycin resistance marker contained in the T-DNA, suggesting that themutation in this line may be a disrupted, tagged allele. dwf4-2 alsocontains a single kanamycin resistance marker, but it failed tocosegregate with the dwarf phenotype. Two additional alleles (dwf4-3 anddwf4-4) were identified among dwarf mutants obtained from the NottinghamArabidopsis Resource Centre (Nottingham, UK; N365 and N374). Unlessotherwise indicated, all experiments presented below were performed withdwf4-1.

[0178] Standard molecular techniques were performed as describedpreviously (Sambrook et al. 1989). The plant DNA flanking the T-DNA wascloned using the plasmid rescue technique as described by Dilkes andFeldmann (1998) “Cloning genes from T-DNA tagged mutants” in Methods inMolecular Biology: Arabidopsis Protocol, J. Martinez-Zapater and J.Salinas, eds (Totowa, N.J.: Humana Press), pp. 339-351. Briefly, dwf4-1genomic DNA was digested with EcOR1 (for the right border) or SalI (forthe left border), ligated under conditions to maximize intramolecularevents, and introduced into competent Escherichia coli cells. Theresulting colonies were screened on ampicillin. Five colonies from theleft border transformation contained plant DNA flanking the insertionsite. The restriction pattern displayed two different types of plantDNA. Three contained a 5.6-kb insert, whereas the other two contained a1.1-kb insert. This result suggested that the T-DNA insert in dwf4-1 wasflanked by two left border sequences. The existence of two left bordersequences was confirmed by gel blot analysis with genomic DNA, using theputative plant flanking DNAs as probes. A single wild-type EcORIfragment was split into two fragments in dwf4-1.

[0179] Wild-type genomic clones were isolated from a library made fromWs-2 DNA by using the 5.6-kb fragment as a probe. The library wasconstructed using X DASH-II arms (Stratagene, La Jolla, Calif.).Approximately 10,000 primary plaques were screened. Duplicate-filterscreening resulted in 12 positives. Restriction mapping of the secondaryclones revealed that some contained part of the DWF4 locus. In fact, oneof the clones, D4G12-1, contained an intact 13-kb DNA spanning the T-DNAinsertion site. The 13-kb insert in D4G12-1 was subcloned intopBluescript SK− (Stratagene). Subclones were sequenced from each end ofthe insert by using the universal primers in the plasmid. DNA sequencingwas performed using an ABI 377 (Perkin-Elmer, Norwalk, Conn.) automatedsequencer at the Arizona Research Laboratories (Tucson, Ariz.).

[0180] Reverse transcriptase-polymerase chain reaction (RT-PCR) was usedto isolate a cDNA clone. RNA was isolated from 5-day-old dark- andlight-grown seedlings. Superscript II reverse transcriptase (BRL,Gaithersburg, Md.) was used for the cDNA synthesis, according to themanufacturer's protocol. Briefly, 7 μg of total RNA was mixed with thereverse primer, D4R3. To the heat-denatured RNA-primer mix, the RTmixture was added and incubated for 1 hr at 43° C. Two microliters of RTproduct was used for PCR amplification by using different primers setsintended to cover all of the putative coding region. RT-PCR productswere fractionated on an 0.8% agarose gel (Sambrook et al. 1989); theexpected bands were purified using a Geneclean kit (BIO 101, Inc.,Vista, Calif.), further amplified, and sequenced to determine the codingregion.

[0181] B. Sequencing

[0182] dwf4-2 was isolated from a T-DNA mutant population as an untaggedallele, whereas dwf4-3 and dwf4-4 were obtained from plants obtainedfrom the Nottingham Arabidopsis Stock Centre (University of Nottingham,UK; stock nos. N365 and N374); the mutagenesis method for these twolines is not known. Based on the DNA sequence of wild-type genomic DNA,pairs of primers were designed to amplify -1-kb stretches of genomicDNA. Oligonucleotide sequences are shown 5′ to 3′. The numbers showncorrespond to positions in the genomic sequence, with the adenine basein the translation initiation codon set as position 1. D40VERF,1-ATGTTCGAAACAGAGCATCATACT-24 (SEQ ID NO:3); D4PRM,(−1)—CCTCGATCAAAGAGAGAGAGA-(−21) (SEQ ID NO:4); D4RTF,143-TTCTTGGTGAAACCATCGGTTATCTTAAA-171 (SEQ ID NO:5); D4RTR,853-TATGATAAGCAGTTCCTGGTAGATTT-828 (SEQ ID NO:6); D4F1,(−242)—CGAGGCAAC-AAAAGTAATGAA-(−222) (SEQ ID NO:7); D4R1,689-GTTAGAAACTCTAAAGATTCA-669 (SEQ ID NO:8); D4F2,576-GATTCTTGGCAACAAAACTCTAT-598 (SEQ ID NO:9); D4R2,1685-CCGAACATCTTTGAGTGCTT-1666 (SEQ ID NO:10); D4F3,1606-GTGTGAAGGTTATAAATGAAACTCTT-1631 (SEQ ID NO:11); D4R3,3156-GGTTTAATAGTGTCGACACTAATA-3132 (SEQ ID NO:12); D4F4,2316-CCGATGACTTGTACGTGCGTTA-2337 (SEQ ID NO:13); D4F5,730-GCGAAGCATATAATGAGTATGGAT-753 (SEQ ID NO:14); and D4R5,1876-GTTGGTCATAACGAGAATTATCCAAA-1851 (SEQ ID NO:15). Because the twostock center lines were in a different genetic background than thewild-type gene that we had sequenced (WS), primers were based primarilyon the exon sequence to avoid sequence variation between introns.Genomic DNA isolated from the mutants was subjected to PCR, using theseprimer sets. The amplified DNA fragments were fractionated on 0.8% TAEagarose gel (Sambrook et al. 1989), purified using Geneclean (BIO 101,Inc.) or Qiaquick™ columns (Qiagen Inc., Chatsworth, Calif.), andsequenced. Putative mutations were identified by comparing the mutantDNA sequence with the wild-type sequence. The sequence was confirmed bysequencing independently amplified fragments at least three times foreach mutation to eliminate PCR misincorporation.

[0183] C. Sequence Analysis

[0184] Annotations in multiple sequence alignment were performed usingthe ALSCRIPT package provided by Barton, G. J. (1993) Protein Eng.6:37-40. Searches for similar protein sequence were performed with theBLAST program (Altschul et al. (1990), supra). In addition, usefulpackages, available on the internet, such as promoter, proteintargeting, polyadenylation site, and splice site, have been employed tocharacterize the DNA and protein sequence (consolidated in the searchlauncher, Baylor College of Medicine, Baylor, Tex.). All other sequenceanalysis was performed using the Genetics Computer Group (Madison, Wis.)software package.

[0185] Analysis of the complete genomic sequence, starting at the EcORIsite, with the promoter prediction by neural network (NNPP) package(http://www-hgc.lbl.gov./projects/promoter.html), indicated that thegene included a putative promoter (TATAT is found in the putativepromoter region between nucleotides −143 to −78) and polyadenylationsignal sequences (AATAA near a position at 3238 bp and a putativeGU-rich signature from 3283 to 3290 bp).

[0186] Unsuccessful attempts to detect mRNA by tissue-specific RNA gelblot analysis, using the 4.8-kb fragment as a probe, suggested that DWF4encoded a rare message. In addition, there were no matching expressedsequence tags in the Arabidopsis database. Therefore, we screened twodifferent cDNA libraries made with either normalized mRNA from differenttissues or RNA from floral tissues, using the 4.8-kb fragment as a probe(ABRC stock numbers CD4-7 and CD4-6, respectively). After finding nopositives in 109 clones screened, we chose to directly amplify DWF4 cDNAfrom total RNA made from 5-day-old seedlings, using reversetranscriptase-polymerase chain reaction (RT-PCR). Whereas RNA from bothlight-grown and dark-grown seedlings yielded the expected RT-PCRproducts, RNA from dark-grown seedlings generated significantly more.The bands were gel purified and sequenced. Alignment of the genomic andcDNA sequences indicated that the DWF4 gene was composed of eight exonsand seven introns (FIG. 2A; FIG. 10).

[0187] Sequence analysis of the dwf4-1 allele revealed that the T-DNAwas inserted in the 5′ end of intron 7 (FIG. 2A). In addition, sequenceanalysis of the left border plant junctions indicated that at onejunction (5′), 75 bp of unknown DNA was inserted, whereas at theotherjunction (3′), 24 bp of left border and 19 bp of plant DNA weredeleted. To prove that DWF4 had been cloned, two other dwf4 alleles(dwf4-2 and dwf4-3) were sequenced to identify possible lesions. Asshown in FIG. 2B, dwf4-2 contained a deletion of three conserved aminoacids (324 to 326) caused by a 9-bp deletion, and dwf4-3 contained apremature stop codon (289) caused by changing a tryptophan codon (UGG)to a nonsense codon (UGA). Due to a premature stop codon, translation ispredicted to be terminated before the heme binding domain, which isessential for cytochrome P450 function (Poulos et al. (1985) J. Biol.Chem. 260:16122-16130). Because T-DNA-generated alleles dwf4-1 anddwf4-2 and an additional mutant allele all possess loss-of-functionmutations affecting the same protein, we conclude that we have clonedthe DWF4 gene.

Example 3 The DWF4 Gene Encodes a Cytochrome P450

[0188] The open reading frame of DWF4 encodes a protein composed of 513amino acids. BLAST database searches (Altschul et al. (1990) J. Mol.Biol. 215:403-410) for similar sequences yielded a superfamily ofcytochrome P450 proteins as significant high-scoring segment pairs.Cytochrome P450s are heme-thiolate enzymes. They display acharacteristic Soret absorption peak at 450 nm when the substrate-bound,reduced form is exposed to the light (Jefcoate (1978) “Measurement ofsubstrate and inhibitor binding to microsomal cytochrome P-450 byoptical-difference spectroscopy” in Methods in Enzymology, Vol. 52, S.Fleischer and L. Packer, eds (London: Academic Press), pp. 258-279).Typical microsomal cytochrome P450s hydroxylate various substrates viatheir monooxygenase activity, which utilizes molecular oxygen andreducing equivalents from NAD(P)H. In addition to the hydroxylation,other activities of cytochrome P450 enzymes, such as oxidation,dealkylation, deamination, dehalogenation, and sulfoxide formation, areinvolved in a variety of biological events in catabolism, anabolism, andxenobiotic metabolism in plants as well as animals (reviewed in West(1980) “Hydroxylases, monooxygenases, and cytochrome P-450” in TheBiochemistry of Plants: A Comprehensive Treatise, Vol. 2, Metabolism andRespiration, D. D. Davies, ed (New York: Academic Press), pp. 317-365;Nebert and Gonzalez (1987), supra; Guengerich (1990) Crit. Rev. Biochem.Mol. Biol. 25:97-152, Guengerich (1993) Am. Sci. 81:440-447; Durst(1991) “Biochemistry and physiology of plant cytochrome P-450” inMicrobial and Plant Cytochromes P-450: Biochemical Characteristics,Genetic Engineering and Practical Implications, K. Ruckpaul and H. Rein,eds (London: Taylor and Francis), pp. 191-232; Bolwell et al. (1994)Phytochemistry 37:1491-1506; Durst and Nelson (1995), supra; Schuler(1996) CRC Crit. Rev. Plant Sci. 15:235-284). Evolutionarily, cytochromeP450s have been found in a broad spectrum of living organisms, and theyshare significant homology at the amino acid sequence level. Thus, ithas been proposed that all known cytochrome P450s were derived from acommon ancestor (Nelson and Strobel (1987) Mol. Biol. Evol. 4:572-593).

[0189] Typical cytochrome P450s contain four characteristic domains asdefined by Kalb and Loper 1988. Of the four domains, A, B, C, and D, atleast two of them have been assigned specific functions. Domain A bindsa substrate and molecular oxygen, and domain D has been shown to bindheme-prosthetic groups via a thiolate bond (Poulos et al. 1985). Thus,typically, microsomal cytochrome P450 enzymes can be identified by theircharacteristic signature sequences, including the heme binding domain,domain A (also referred to as dioxygen binding), domain B (steroidbinding), and domain C (Nebert and Gonzalez (1987) Annu. Rev. Biochem.56:945-993; Kalb and Loper (1988) Proc. Natl. Acad. Sci. USA85:7221-7225). All of these signature sequences were found in DWF4; therelative positions of the domains are indicated in FIG. 2B.

[0190] Durst and Nelson (1995) Drug Metab. Drug Interact. 12:189-206classified plant cytochrome P450s into two distinct groups based ontheir clustering nature in a phylogenetic tree. All of the group Afamilies cluster and are assumed to originate from a common plant P450ancestor. The group A cytochrome P450s conform to the characteristicconsensus sequences (A/G)GX(D/E)T(T/S) in domain A (also called helix I)and PFG(A/S/V)GRRXC(P/A/V)G of the heme binding domain (D) with only afew exceptions. Group A cytochrome P450s appear to catalyzeplant-specific reactions such as lignin biosynthesis (FIG. 6; GenBankaccession number P48421). By contrast, P450s that do not belong to groupA (non-A P450s) are scattered in the phylogenetic tree. They share moreamino acid identity/similarity with P450s found in animals, microbes,and fungi than with those found in plants. The non-A P450s possessfunctions, such as steroid metabolism, that are not limited to plants.Generally, non-A P450s have limited homology with known domainsdescribed for group A.

[0191] The most similar protein to DWF4 is the Arabidopsis CPD protein,a non-A P450. A mutation in CPD also caused dwarfism (Szekeres et al.1996; CYP90A1, GenBank accession number X87368). DWF4 and CPD share 43%identity and 66% similarity. Conforming to the recommended nomenclaturefor cytochrome P450 enzymes, DWF4 and CPD (CYP90A1) are grouped into thesame family within different subgroups (Durst and Nelson (1995) DrugMetab. Drug Interact. 12:189-206). As such, DWF4 represents a secondmember of the CYP90 family and is designated CYP90B1. Sequencesimilarity between the two proteins occurs throughout their length, withthe greatest similarity in the classically conserved domains. Residuesconserved between DWF4 and CYP90A are boxed and italicized in FIG. 3.The second most similar protein is the tomato CYP85 (Bishop et al.(1996), supra; GenBank accession number U54770). A mutation in this genealso results in dwarfism. DWF4 and CYP85 share 35% identity and 59%similarity in their overall protein sequences.

[0192] Six cytochrome P450 sequences with the greatest homology to DWF4,CYP90A1, CYP85, CYP88 (Winkler and Helentjaris (1995) Plant Cell7:1307-1317; GenBank accession number U32579), cyanobacteria CYP120(Kaneko et al. (1996) DNA Res. 3:109-136; GenBank accession numberD64003), human CYP3A3X (Molowa et al. (1986) Proc. Natl. Acad. Sci. USA83:5311-5315; GenBank accession number M13785), and zebrafish CYP26(White et al. White (1996) J. Biol. Chem. 271:29922-29927; GenBankaccession number U68234), were chosen for multiple sequence alignment.Putative domains defined by Kalb and Loper (1988), supra are boxed andlabeled in FIG. 3. First, the heme binding domain pFGgFpRlCpGkel matchescompletely the sequence defined previously. Uppercase letters in thedomain indicate amino acids conserved at all seven sequences in thealignment, and lower-case letters represent residues conserved in atleast half of the proteins. Of the amino acids conserved in the hemebinding domain, the function of the cysteinyl is established as athiolate ligand to the heme (Poulos et al. (1985), supra).

[0193] Domain A is defined by xllfaGhEttssxIxxa. Lowercase x's indicatevariable amino acids. An invariant glutamate (E) preceded threonine (T)at position 314, T314, which is believed to bind dioxygen, was conservedin all proteins compared except CYP88 of maize. The second signaturesequence, domain B, is also conserved in DWF4 with significantsimilarity. A valine at position 370 is conserved in all of theproteins, but it does not appear in Kalb and Loper's classic report(1988) on conserved domains. Again, DWF4 matches the domain C consensussequence. Finally, the anchoring domain in the N-terminal end wasdistinguished by a repeat of the hydrophobic residue leucine. Inaddition, in DWF4, two acidic (glutamate) and two basic (histidine)residues precede the repeated leucine in the N-terminal leader sequence.These charged residues may add more stability to the membrane topologyof the protein as a strong start-stop transfer peptide (von Heijne(1988) Biochim. Biophys. Acta 947:307-333).

[0194] Thus, phylogenetic analyses of these seven proteins withcytochrome P450s unique to plants (group A; Durst and Nelson (1995),supra) indicate that DWF4 does not cluster with these cytochrome P450s(FIG. 6). Rather, DWF4 clustered with cytochrome P450s from otherorganisms: cyanobacteria (CYP120), rat (CYP3A2), human (CYP3A3X), andplants (CYP90, CYP85, and CYP88). DWF4 also deviates from the consensussequence in the group A heme binding domain in that it possesses aPFGGGPRLCAG sequence in which arginine (R) is substituted for proline(P). However, domain A of DWF4, AGHETS, fits the consensus of domain Aof group A. These characteristics suggest that DWF4 is a monooxygenase,similar to P450s of group A, that utilizes molecular oxygen as a sourceof the hydroxyl group, but it mediates some reaction(s) that are notnecessarily specific for plants, for instance, steroid hormonebiosynthesis, which is a critical event for animals. In fact, thesimilarity of DWF4 to the rat testosterone 6β-hydroxylase (34%; GenBankaccession number 631895) or glucocorticoid-inducible hydroxylase (31%;Molowa et al. 1986; GenBank accession number M13785) supports this idea.Further, the similarity that DWF4 shares with CYP90A and CYP85, 66 and59%, respectively, is additional proof that it is involved in plantsteroid biosynthesis (Bishop et al. 1996; Szekeres et al. 1996).

Example 4 The dwf4 Phenotype

[0195] As formally defined, a plant with a dwarf phenotype is one thathas a short, robust stem and short, dark green leaves. dwf4 mutants aresignificantly smaller than the wild type and are dark green in color.They have short, rounded leaves. Again, the dwf4 phenotype isreminiscent of the light-regulatory mutant det2 (Chory et al., supra);however, complementation analysis has shown that the two mutations arenot allelic, with the dwf4 mutation mapping to the lower arm ofchromosome 3 and det2 mapping to chromosome 2 (Chory et al., supra). Theresults presented in Table 1 show that soil-grown dwf4 plants attained aheight of <3 cm at 5 weeks, whereas wild-type plants grew to >25 cm.Moreover, individual organs, such as leaves, were invariably shorter indwarf plants. dwf4 siliques were also markedly shorter than those of thewild type and were infertile. The loss of fertility of dwf4 was due tothe reduced length of the stamen filaments relative to the gynoecium,which resulted in mature pollen deposition on the ovary wall rather thanon the stigmatic surface. Hand pollination of dwf4 flowers with eithermutant or wild-type pollen resulted in good seed set withoutsignificantly changing the size of the siliques. TABLE 1 The Developmentof Wild-Type and dwf4-1 plants Measurement Wild-Type^(a) dwf4-1^(a) FiveWeeks Height  25.8 ± 2.6 cm  2.8 ± 0.3 cm Leaf blade length^(b)  1.72 ±0.36 cm  0.96 ± 0.15 cm Leaf blade width^(b)  0.77 ± 0.10 cm  0.99 ±0.18 No. inflorescences  3.6 ± 0.5  10.5 ± 1.4 No. rosettes  7.1 ± 0.9 13.5 ± 1.3 Other start of flowering  21.5 days  25.9 days maturesilique  1.16 ± 0.07 cm  0.29 ± 0 cm length No. seeds per  37.7 ± 3.3 0.0 silique Final no. of siliques 336.5 ± 90.6 988.4 ± 214.2 Height atmaturity  27.0 ± 2.7  11.6 ± 1.0 cm

[0196] Another feature of dwf4 plants is a reduction in apicaldominance, as was evident by the threefold increase in the number ofinflorescences at 5 weeks of age (Table 1). Mutants also had twice thenumber of rosette leaves, which may be explained by a prolongedvegetative phase in the dwf4 plants. Development of flowers on theprimary inflorescence was delayed by ˜4 days in dwf4, but the floweringphase was significantly longer in the mutant, with senescence of thelast flower occurring at 98 days compared with ˜57 days for the wildtype. One result of this delay in senescence was that dwf4 plantscontained almost three times the number of siliques as did the wild type(Table 1).

[0197] The reduced stature observed in soil-grown dwf4 was also observedin hypocotyls of agar-grown seedlings. Measurements of hypocotyl lengthover time indicated that not only were dwf4 seedlings shorter thanwild-type seedlings immediately after germination but also that the rateof growth was retarded in the mutants (FIG. 5). In addition, dwf4hypocotyls reached their terminal length in<5 days, whereas wild-typeseedlings continued to grow.

[0198] In sum, the dwf4 phenotype can be described as being due to bothprimary and secondary effects of reduced cell elongation. The primaryeffect is simply a reduction in the length of individual organsexclusively along their normal growth axis; that is, organ width is notreduced (Table 1). The secondary effects of reduced cell elongation arethemselves due to the reduction in organ length. The dark green color ofthe leaves, for example, may be due exclusively to the existence of awild-type number of chloroplasts in a significantly smaller cell.Similarly, the sterility of mutants is a consequence of the shortness ofthe stamens, which fail to deposit their pollen on the stigmaticsurface. In addition to the morphological alterations of dwf4, mutantsdisplay delayed development, the first sign of which occurs at flowering(Table 1). Because rosette leaves are produced continuously duringvegetative development, delayed flowering results in dwf4 rosetteshaving almost twice the number of leaves observed in the wild type.

Example 5 The Growth Defect of dwf4 is Due to a Reduction in Cell Length

[0199] Both the short stature and the reduced growth rate of dwf4 couldbe due to a defect in cell division or cell elongation or both. Todistinguish between these possibilities, we analyzed sections from7-day-old hypocotyls and 5-week-old inflorescence stems, by lightmicroscopy, as described in Example 1. To minimize variations due to thedevelopmental stage of the sample, we always took the stem sections fromthe fourth internode. As shown in Table 2, the average cell size in dwf4is significantly smaller than in wild-type plants, whereas nodifferences were detected in the number of cells along the length ofeither organ between the wild type and dwf4. Therefore, the shortstature and reduced organ length of dwf4 are largely or exclusively dueto a failure of individual cells to elongate. No differences wereobserved in the number of cell layers contained in the wild type anddwf4. TABLE 2 Cell Length in Wild-Type and dwf4 plants MeasurementWild-type dwf4 average cell length in hypocotyl: 7 day old 92.7 μm 32.2μm plant average cell length in stem: 5-week old plant 79.2 μm 15.0 μm

[0200] The small size of dwf4 cells offers a possible explanation forthe dark green color of the mutant plants. Chlorophyll measurements weretaken, and leaf mesophyll protoplasts were prepared, stained, andmeasured to visualize and count chloroplasts, as described in Methods.Although there were no significant differences in total chlorophyllcontent, the chlorophyll a/b ratio, or the absorption spectra betweenwild-type plants and mutants, the mean plane area (the apparenttwo-dimensional surface area of mounted cells) of dwf4 leaf mesophyllprotoplasts was 376 mm², whereas that of wild-type protoplasts was 599mm². The two-dimensional comparison of plane area represents a dramaticreduction in volume for dwf4 cells. However, the number of chloroplastsper cell was only slightly lower: the mean number of chloroplasts percell was 40 for dwf4 and 44 for the wild type. Therefore, dwf4 cellscontain a greatly increased number of chloroplasts per unit cell volume.As a consequence, the chloroplasts are brought closer to each other,making the color of the leaves appear darker. Chloroplast size was thesame in both lines.

[0201] Thus, the rate of growth was significantly reduced in agar-growndwf4 seedlings, which ceased to grow when their hypocotyl length was<20% of the final wild-type length. Because all of the cells in ahypocotyl before the initiation of leaf development are present in theembryo, the initial growth of seedlings is due exclusively to cellexpansion, which therefore must be reduced in dwf4. A similar situationapplies to soil-grown plants. Five weeks after germination, well afterplants had bolted, dwf4 plants were shorter than wild-type plants (Table1). Although the mutants continued growing for several weeks more thandid the wild type, they remained shorter through senescence. That cellelongation is the direct cause of this decreased growth is shown bymeasurements of cell length both in 7-day-old hypocotyls (Table 2) andin 5-week-old stems (Table 2). Not only is the reduction in cell lengthin good agreement with the reduction in organ length, but insofar ascould be determined, there is no difference in the number of cellsbetween dwf4 and wild-type plants.

[0202] Organ growth by cell elongation in plants occurs as part ofnormal development in response to a variety of input signals. Mutantsthat are defective in these signaling pathways invariably fail toelongate normally in response to the appropriate stimuli. A mutant witha block at a step that is common to several individual pathways wouldtherefore be expected to have defective responses to all of thecorresponding signals. dwf4 appears to be such a mutant. FIG. 6 showsthat elongation induced by the hy2 mutation is blocked in a dwf4 hy2double mutant. Not surprisingly, in view of this result, dwf4 alsofailed to display hypocotyl elongation as a response to growth incomplete darkness. In addition, dwf4 was capable of perceiving GA, butits response was severely compromised. This mutant could also respond tothe inhibitory effects of auxin but was incapable of auxin-stimulatedelongation. It was only exogenous BL that fully restored wild-typelength to dwf4 hypocotyls (Choe et al. (1998), supra).

[0203] Because dwf4 failed to respond to at least three independentsignaling pathways but responded fully to only one, the most likelyexplanation for the dwarf phenotype is therefore that a fully functionalBR system is required for a full response to GA, auxin, anddeetiolation. From the perspective of cellular economy, it may beadvantageous that the downstream elements involved in cell elongationare shared among at least some of the signaling pathways that evoke thisresponse. The interaction of various pathways at a common step providesthe plant with a potential point for the integration of signals producedby diverse independent stimuli. Our results indicate that BRs act atthis downstream step.

Example 6 dwf4 is Specifically Rescued by Brs

[0204] The reduced length of cells in dwf4 hypocotyls and inflorescencestems is indicative of a failure of these cells to elongate duringdevelopment. A variety of endogenous and environmental signals isresponsible for stimulating elongation in plants; therefore, a series ofexperiments was performed to determine whether dwf4 is affected in aspecific signaling pathway or is blocked in elongation as a response tovarious signals.

[0205] Of the endogenous (hormonal) signals that might be deficient indwarf plants, an obvious candidate is GA, because gibberellin-deficientmutants are shorter in stature than are the wild-type plants (Koornneefand Van der Veen, supra). Our results, however, indicate that dwf4 isnot defective in the synthesis of gibberellins. When germinated on 10⁻⁵M GA, wild-type seedlings demonstrated an elongation response (FIG. 6),whereas dwf4 seedlings responded minimally, if at all. At 10⁻⁴ M GA,wild-type seedlings elongated slightly more than at 10⁻⁵ M, but the dwf4seedlings were essentially saturated for elongation at 10⁻⁵ M GA.Similar results were obtained when soil-grown plants were sprayed with 1mM GA once inflorescences first became visible: dwf4 inflorescence stemselongated by only 28% above the untreated controls, whereas those of thewild type elongated by 45% above the untreated controls. Mutants thatowe their reduced stature to decreased levels of endogenous gibberellinscan be fully rescued by added hormone (Koornneef and Van der Veen,supra; Talon et al., supra). In addition, dwf4 seeds germinate in theabsence of exogenously supplied GA. Our results therefore suggest thatdwf4 is not deficient in endogenous GA. A corollary conclusion from thisexperiment is the demonstration that dwf4 is capable of detecting GA;that is, it is not likely to be affected in signal perception but ratheris defective in the extent to which it can respond to this signal.

[0206] Auxin can also stimulate cell elongation. This effect isespecially visible in young seedlings (Klee and Estelle, supra). Theresponse of wild-type and dwf4 plants to auxin was tested by growingseedlings for 10 days on vertically oriented plates containing variousconcentrations of the synthetic auxin 2,4-D. At all concentrationsassayed, inhibition of root growth was evident. FIG. 6 shows that at10⁻⁸ M 2,4-D, hypocotyl elongation in wild-type and dwf4 seedlings wassimilar to that of the controls. Higher concentrations of auxin wereinhibitory for both wild-type and dwf4 seedlings, and lowerconcentrations had no effect. In view of the inhibition of root growth,it is clear that dwf4 is not auxin resistant; rather, its elongationresponse is compromised.

[0207] As mentioned above, the most obvious exogenous signal for plantsis light. Therefore, to investigate whether light-regulated cellelongation is altered in dwf4, wild-type and dwf4 seedlings were grownin the dark, as described in Example 1. FIG. 6 shows that as expected,wild-type seedlings displayed hypocotyl elongation typical of etiolatedgrowth. By contrast, dark-grown dwf4 seedlings were only slightly longerthan those grown in the light. To assess the relationship between thedwf4-phenotype and light sensing by dwf4, the mutation was crossed intoa mutant defective in the HY2 gene. All hy mutants share the commonphenotype of an elongated hypocotyl that mimics part of the etiolationresponse in the light. Specifically, hy2 is deficient in activephytochrome because chromophore biosynthesis does not take place (Choryet al. (1989a) Plant Cell 1:867-880). FIG. 6 shows that dwf4 hy2 doublemutants displayed a dwarfed phenotype indistinguishable from that ofdwf4 HY2 (light-grown control); therefore, the elongation block due tothe dwf4 mutation is epistatic to a defect in phytochrome activity.

[0208] In the course of our studies, we prepared a genomic library fromdwf4-1, from which we isolated a clone in which a fragment of T-DNAinterrupts a gene encoding a putative cytochrome P450 steroidhydroxylase. Because BRs have been shown to elicit elongation inArabidopsis (Clouse et al. (1993) J. Plant Growth Regul. 12:61-66) andbecause BR-deficient mutants have been recently described (Kauschmann etal. (1996), supra.; Li et al. (1996), supra; Szekeres et al., supra), wetested the effect of BL on Arabidopsis seedlings by germinating seeds inliquid medium containing different amounts of BL. As shown in FIG. 6,the dwf4 hypocotyls were restored to wild-type height by 10⁻⁶ M BL.This, together with our identification of a disrupted gene encoding aputative BR biosynthetic enzyme, strongly suggests that the phenotype ofdwf4 is specifically due to a defect in BR biosynthesis (see Choe et al.(1998) Plant Cell 10:231-243).

[0209] Thus, the results indicate that BL is involved at or near adownstream control point where multiple signaling pathways interact.First, as shown in FIG. 6, BL is required for cell elongation as aresponse to darkness as well as GA and auxin. In addition, previousstudies (Kauschmann et al. (1996), supra; Li et al. (1996), supra;Szekeres et al. (1996), supra) and the work described herein show thatBR can compensate for the cell elongation defect of mutants as diverseas det2, cpd, dwf4, det1, cop1, and dwf1. This places BRs downstream ofall the cellular functions affected in these mutants. Finally, at leastone of the BR biosynthetic genes has been shown to be modulated bylight, cytokinins, and the carbon source (Szekeres et al. (1996),supra).

[0210] Mutations in axr2 result in a dwarf growth habit and a dark-grownphenotype with short hypocotyl and open cotyledons (Timpte et al.(1992), supra). In addition, axr2 mutants are resistant to auxin,ethylene, and abscisic acid and have defective root and shootgravitropism. The dwarf phenotype in axr2 mutants has been shown to bedue to reduced cell elongation and is rescued by BL (Szekeres et al.(1996), supra). This suggests that at least one of the multiple hormonesignaling pathways affected in axr2 involves a BR-dependent step.Mutations at another locus, acaulis1, also have a significant reductionin cell elongation, but the defect is confined to inflorescence stemsand leaves (Tsukaya et al. (1993) Development 118:751-764). Flowers arefully fertile and mature into normal-sized siliques with normal seedset. There is no change in hypocotyl length. If BRs are directlyinvolved in this apparently organ-specific signaling pathway, it may bedue to organ-specific responsiveness to individual BR species. Withregard to the mechanism of action of BRs, at the moment one can onlyspeculate that the target may be a component of the cell expansionmachinery. Perhaps steroid signaling initiates a series of eventsleading to cell wall loosening.

Example 7 The Elongation Defect of dwf4 Leads to a Light-RegulatoryPhenotype

[0211] The BR-deficient mutant det2 was originally identified asdefective in regulation by light (Chory et al. (1991), supra). Given thesimilarity of det2 and dwf4 phenotypes and functions and in view of theobservation that dwf4 is epistatic to hy2, one can predict that theetiolation response, which includes significant hypocotyl elongation,would not be normal in dwf4. To assess to what extent the etiolationresponse is affected by BR-dependent cell elongation, we grew dwf4 andwild-type plants on agar under continuous light or in complete darkness,as described above in Example 1. After 7 days of growth in the light,wild-type seedlings displayed open and expanded cotyledons as well asemerging leaf buds. In contrast, the overall appearance of light-growndwf4 seedlings was strikingly similar to that of det2 (Chory et al.(1991), supra). dwf4 hypocotyls were very short, and the cotyledons weresmaller than those of the wild type, displaying significant epinasticgrowth. As expected, dark-grown wild-type seedlings had a typicaletiolated appearance, with a highly elongated hypocotyl and closed,unexpanded cotyledons. However, dwf4 hypocotyls failed to elongate. Thatthe dwf4 mutation can abolish the elongation component of the etiolationresponse is in agreement with the notion that the block in cellelongation in dwf4 is specifically a BR-dependent process.

[0212] In addition to short hypocotyls, dark-grown dwf4 seedlingsdisplayed partially open cotyledons and leaf primordia, with up to fourleaf buds clearly visible. This has not been observed with the wildtype, although it occurs with certain light-regulatory mutants (Chory etal. (1989b), supra; Deng et al. (1991) Genes Dev. 5:1172-1182; Wei andDeng (1992), supra). dwf4 leaf development continued in the darkness forseveral weeks, resulting in significant expansion of rosette leaves.These results indicate that dwf4 plants can initiate what is normally aphotomorphogenic pathway in the absence of light. Although this is oftendiagnostic of a light-regulatory mutant, wild-type Arabidopsis canperform leaf development and even flowering in complete darkness whengrown in liquid culture (Araki and Komeda (1993) Plant J. 4:801-811).

[0213] The cause for this dark-flowering effect is not understood;therefore, the possibility exists that leaf development in dark-growndwf4 is related to dark flowering and not to a light-regulatory defect.For example, perhaps the proximity of the dwf4 shoot apical meristem tothe surface of the agar, due to the shortness of the hypocotyls, mimicssome effect of submerged culture, such as a high water potential or ahigh concentration of some nutrient. To test this possibility, wild-typeseedlings were grown in complete darkness for 6 weeks in verticallyoriented dishes to maximize contact between the seedling and the medium.Wild-type seedlings grown in this fashion displayed open cotyledons andunderwent at least partial leaf development. In fact, all wild-typeseedlings grown along the surface of the agar showed development of aninflorescence with at least one cauline leaf and a terminal flower bud.We conclude, therefore, that the appearance of leaves in dark-grown dwf4may be due simply to its short size and the culture conditions.

[0214] A number of light-regulatory mutants have been described thatundergo photomorphogenesis in the dark at the cellular level. In mutantssuch as cop1, cop8, cop9, cop10, and cop11, stomata undergophotomorphogenic maturation (Deng and Quail (1992), supra; Wei and Deng(1992), supra; Wei et al. (1994), supra); of these, cop1 and cop9 aswell as det1 (Chory et al. (1989b), supra) also initiate differentiationof plastids into chloroplasts. To determine whether dwf4 plants undergophotomorphogenic cellular differentiation in the dark, we analyzedcotyledons from light- and dark-grown plants by transmission andscanning electron microscopy. Analysis of plastids in thin sections from7-day-old dark-grown seedlings showed no difference between the wildtype and dwf4. Both lines contained normal chloroplasts when grown inthe light, whereas dark-grown seedlings contained etioplasts, with theircharacteristic prolamellar body and no significant organization ofthylakoids. Analysis of stomatal structures on the underside ofcotyledons from 7-day-old seedlings indicates that stomatal developmentwas not completed in the dark, because the stomatal opening was occludedin both lines. The majority of light-regulatory mutants analyzed to datedisplayed light-grown morphology in the dark without concomitantchloroplast or stomatal development. As in these mutants, therefore, thedwf4 mutation uncouples the developmental pathway of seedling morphologyfrom that of light-regulated cellular differentiation.

[0215] An additional feature of many light-regulatory mutants is thatphotomorphogenesis in the dark is accompanied by expression of genesthat normally are light induced (Chory et al. (1989b), supra, Chory etal. (1991), supra; Deng et al. (1991), supra; Wei and Deng (1992),supra; Hou et al. (1993), supra; Wei et al. (1994), supra). To assesswhether dwf4 is able to induce light-regulated transcripts in the dark,we compared the activity of a CAB promoter fused to the Escherichia coligene uidA, encoding β-glucuronidase (GUS), in light- and dark-growndwarf and wild-type plants. The CAB-uidA fusion in pOCA107-2 (Li et al.(1994) Genes Dev. 8:339-349) was crossed into dwf4, and F2 dwarf andwild-type plants were grown in the dark or light for 12 days, followedby determination of GUS activity by fluorometry (Gallagher (1992).“Quantitation of GUS activity by fluorometry” in GUS Protocols, S. R.Gallagher, ed (New York: Academic Press), pp. 47-59).

[0216] The results demonstrated that when grown in the light, bothwild-type and dwf4 seedlings contained GUS activity, which wassignificantly reduced in both lines when grown in the dark. Moreover,dark-grown dwf4 seedlings displayed no GUS activity above the backgroundpresent in dark-grown wild-type plants. The absence of light-inducedgene expression in the dark is a distinguishing feature of certain copand det mutants, such as cop2, cop3, and det3. Because we have shownthat the defect in cell elongation of dwf4 is specifically rescued byBRs, even in the presence of light, we conclude that this is not alight-regulatory mutant. That its phenotype is partially deetiolated orconstitutively photomorphogenic is a secondary effect of its reducedstature and the growth conditions.

Example 8 Abnormal Skotomorphogenesis as a Consequence of the DwarfGrowth Habitat

[0217] When dwf4 is grown in the light, its morphology is similar tothat of various cop and det mutants, with multiple short stems, shortrounded leaves, loss of fertility due to reduced stamen length, anddelayed development (FIG. 6). Dark-grown dwf4 seedlings possess shorthypocotyls, open cotyledons, and developing leaves. Therefore, it istempting to speculate that this mutant may be defective in the controlof light-regulated processes. On the other hand, because adark-flowering phenotype has been demonstrated for liquid-grownArabidopsis (Araki and Komeda (1993), supra), and given that agar mediumis mostly water, it is especially significant that it is the dwarfseedlings, whose apical meristems are very close to the agar surface,that display a light-grown phenotype in the dark. Furthermore, becausewild-type seedlings grown along the surface of the agar reproduce thedark-flowering phenotype, it is possible that the apparentlight-regulatory defect of dwarf seedlings is a dark-flowering response.This possibility is strengthened by the observation that wild-typeseedlings (ecotype Wassilewskija [Ws-2]) grown in the dark onhorizontally oriented plates occasionally bend down and touch the agarsurface, and these seedlings invariably produce leaves.

[0218] In addition, of the eight DWF loci identified in this laboratory,only the shortest mutants displayed open cotyledons and leaf buddevelopment; in the case of dwfl (Feldmann et al. (1989), supra), thisaberrant skotomorphogenesis is confined to the most severely affectedalleles. In addition to the presence of a short hypocotyl and at leastpartially open cotyledons in the dark, cop1 (Deng and Quail (1992),supra), detl (Chory et al. (1989b), supra), and det3 (Cabrera y Poch etal. (1993) Plant J. 4:671-682) have been shown to initiate leafformation in the dark. In mutants such as cop1, cop8, cop9, cop10, andcop11, stomata undergo photomorphogenic maturation (Deng and Quail(1992), supra; Wei and Deng (1992), supra; Wei et al. (1994)), supra);of these, cop1 and cop9 as well as det1 (Chory et al. (1989b), supra)also initiate differentiation of plastids into chloroplasts. dwf4displayed, in addition to a light-grown dwarf phenotype, a dark-growthphenotype of short hypocotyls, open cotyledons, and developing leaves;however, in contrast with the light-regulatory defect seen with wholeplants, the cellular differentiation phenotype was unaffected. Indark-grown dwarf seedlings, stomata did not complete their development,and differentiation of chloroplasts was not observed. The absence of acellular light-regulatory phenotype in dwf4 is similar to that of anumber of photomorphogenic mutants, such as det2, det3, cop2, cop3, andcop4 (Chory et al. (1991), supra; Cabrera y Poch et al. (1993), supra;Hou et al. (1993), supra).

[0219] In view of the dark-flowering phenotype on agar and the absenceof a light-regulatory defect in differentiating cells, we conclude thatat least in the case of dwf4, aberrant skotomorphogenesis may be aconsequence of a dwarf growth habit rather than dwarfism being part of adefect in the control of light-regulated processes. This effect may alsoexplain the light-regulatory phenotype found in other mutants withseverely reduced height, such as axr2 (Timpte et al. (1992), supra), andstrong alleles of dwfl, both of which are also rescued by exogenous BRs(Szekeres et al. (1996), supra).

Example 9 Feeding Experiments with BR Biosynthetic Intermediates

[0220] In view of the results described above, we hypothesized that DWF4mediates one or more of several steroid hydroxylation steps in the BRbiosynthetic pathway. To test this, dwf4 was grown on all of theavailable biosynthetic intermediates in the BR biosynthetic pathways andexamined to ascertain which intermediates could rescue the dwarfphenotype. In addition to the intermediates belonging to the early C-6oxidation and late C-6 oxidation pathways (Choi et al. (1997), supra),22α-hydroxycampesterol (22-OHCR), 6α-hydroxycathasterone (6-OHCT)(Takatsuto et al. (1997) J. Chem. Res. (synop.) 11:418-419), and6α-hydroxycastasterone (6-OHCS) (S. Takatsuto, T. Watanabe, T. Noguchi,and S. Fujioka, unpublished data) were synthesized and tested.

[0221] Germinated seedlings were transferred to media supplemented withone of the intermediates or BL to pinpoint the step catalyzed by DWF4.Cathasterone (CT; early C-6 oxidation pathway), 6-OHCT,6-deoxocathasterone (6-deoxoCT; late C-6 oxidation pathway), and22-OHCR, and all of the downstream compounds belonging to each branch,rescued the light-grown dwf4 phenotype, whereas the known precursorsfailed to cause an elongation response. Rescued seedlings exhibitedgreatly elongated cotyledonary petioles and expanded cotyledons,moderately elongated hypocotyls, and leaves that were larger and not ascurled compared with nonrescued dwarfs. In addition, the rescuedseedlings were less green than the dwarfs. These experiments wereconducted in liquid media. Feeding experiments performed in the darkyielded similar results.

[0222] Dose-response tests on the putative substrates and products ofDWF4 were also performed. dwf4 seedlings failed to respond to6-oxocampestanol (6-oxoCN) even at high concentrations (3×10⁻⁶ M).However, on CT the overall morphology of dwf4 was essentially rescued towild-type phenotype at 3×10⁻⁷ M and higher, whereas with 6-deoxoCT,rescue occurred with as little as 10⁻⁷ M and may have even beeninhibitory at higher concentrations. Of particular interest is the moredramatic response of the epicotyls versus the smaller response of thehypocotyls to CT. This same phenomenon was true for seedlings treatedwith >10⁻⁷ M 6-deoxoCT. At concentrations >10⁻⁷ M, the seedlingsdisplayed an inhibition in hypocotyl and root elongation as well ascotyledon and leaf expansion.

[0223] In a dose-response experiment performed in the dark, theseedlings failed to respond to 6-oxoCN (10⁻⁸ to 3×10⁻⁶ M). A higherconcentration of CT for dark-grown seedlings, compared with light-grownseedlings, 3×10⁻⁶ M (FIG. 5B), was required to convert the hypocotyl toa length similar to that of the wild type. High concentrations of6-deoxoCT caused dramatic elongation but were less effective at rescuingdwf4 hypocotyls to wild-type phenotype.

[0224] To determine whether the results of the seedling feedingexperiments could be applicable to soil-grown mature plants, 6-week-olddwf4 plants were treated with BR intermediates and BL. Concentrations ofapplied intermediates were adjusted empirically to optimize responses.Consistent with the results obtained from the seedling experiments, only22α-hydroxylated compounds can rescue the dwf4 phenotype. The elongationresponse was only observed in the young tissues of the inflorescence,regardless of whether the BRs were applied locally or sprayed over theentire plant. In contrast to the striking elongation of the pedunclesand pedicels, fertility was not restored by BR treatment. The sterilityin dwf4 is hypothesized to be mechanical, which means that the filamentsare shorter than the carpels such that the pollen is shed onto the ovarywalls rather than onto the stigmatic surface. In fact, if dwf4 plantsare hand pollinated using dwf4 pollen, fertility increases.

[0225] Pedicels displayed a more consistent response to exogenouslyapplied BRs than did internodes, which led us to quantify thesensitivity of pedicels to these compounds. As shown in FIG. 7, dwf4pedicels were more sensitive to BR intermediates belonging to the lateC-6 oxidation pathway, 6-deoxoCT (10⁻⁶ M) and 6-deoxoteasterone(6-deoxoTE; 10⁻⁶ M), compared with CT (10⁻⁵ M) and teasterone (TE; 10⁻⁵M) of the early C-6 oxidation pathway. The end product of the BRpathway, BL (10⁻⁷ M), possessed the highest bioactivity. Thisconcentration induced approximately the same degree of response as itsprecursor compounds at 10⁻⁶ M. Finally, application of 22-OHCR (10⁻⁵ M)also resulted in a dramatic elongation response (FIG. 7).

[0226] Rescue of dwf4 by 22-hydroxylated steroids confirms that themissing step in dwf4 is hydroxylation at the C-22 position. In fact, wefound that the chemically synthesized 22-OHCR was also effective inrescuing dwf4 (FIG. 7) hydroxylation at C-22. These results indicatethat there is no defect other than 22α-hydroxylation in dwf4 plants.

[0227] In BR biosynthesis, Fujioka and Sakurai (1997b), supra havedemonstrated that there are at least two branched biochemical pathwaysto the end product BL (FIG. 1; Fujioka and Sakurai (1997a), supra,Fujioka and Sakurai (1997b), supra; Sakurai and Fujioka (1997), supra).Depending on the oxidation state of C-6, they are referred to as theearly or late C-6 oxidation pathways. In the early pathway, the C-6 isoxidized to a ketone at canpestanol (CN), whereas in the late pathway itis oxidized at 6-deoxocastasterone (6-deoxoCS). Otherwise, the twopathways share equivalent reactions. Our results from the experimentswith the available BR intermediates clearly demonstrate that dwf4 isdefective in the 22α-hydroxylation steps in each of the pathways.Application of all 22α-hydroxylated intermediates in these pathways,such as CT and 6-deoxoCT, cause dramatic elongation of dwf4 plants, butcompounds not hydroxylated at C-22 had no effect. This result alsosuggests that DWF4 recognizes at least two substrates: CN and 6-oxoCN.It seems reasonable to hypothesize that the same result will be foundfor CPD, a 23α-hydroxylase; that is, it will use 6-deoxoCT as well as CTas substrate.

[0228] The rescue of dwf4 by 22-OHCR is an important observation. First,it confirms DWF4 as a 22α-hydroxylase. Second, this result suggests that22-OHCR was metabolized to induce the same responses as othercomplementing BRs. This is not just a general effect because ourunpublished data show that another dwarf mutant that we have identifiedin our screens, dwf8-1, is not rescued by this compound. Finally, thesefeeding experiments suggest that the metabolism of 22-OHCR may representa new subpathway in the BR biosynthetic pathway. If this compound alsoexists in vivo and constitutes the first step in a separate subpathway,by analogy to the chemical structure, the C-6 hydroxylated BRs, forexample, 6-OHCT, 6-hydroxyteasterone, and so on, may be possibleintermediates in this network. If so, the intermediates in this pathwaymay play a role as bridging molecules between the early and late C-6oxidation pathways. Alternatively, it might be possible that 22-OHCRmerges into one of the two pathways to be metabolized. In this case, thelate C-6 oxidation pathway is the best candidate; our unpublished datashow that 22-OHCR is more effective in the light in rescuing the dwf4phenotype, which is true for all of the intermediates in the late C-6oxidation pathway.

[0229] Currently, biochemical feeding studies suggest that the twopathways merge to produce BL or CS (Yokota et al. (1991), Metabolism andbiosynthesis of brassinosteroids. In Brassinosteroids: Chemistry,Bioactivity, and Application, H. G. Cutler, T. Yokota, and G. Adam, eds(Washington, D.C.: American Chemical Society), pp. 86-96; Yokota, et al.(1997) Plant Physiol. 115(suppl.): 169; FIG. 1). Several lines ofevidence indicate that seemingly redundant pathways can be utilized torespond to environmental or developmental signals. First, the pathwayscould respond to specific signals. For instance, it is possible thatvarious cues such as light, dark, or developmental signals play a rolein regulating these subpathways. Our feeding experiments consistentlyshowed that BRs in the late C-6 oxidation pathway are more effective atpromoting cell elongation in light-grown plants (dwf4 and wild type;FIG. 7) and that the BRs belonging to the early C-6 oxidation pathwayare more active in dark-grown seedlings. Thus, it may be possible thatthe late C-6 oxidation pathway operates in the light and that the earlyC-6 oxidation pathway functions primarily in the dark. Second, ratherthan a simple merger of branched pathways to BL as an end product, eachintermediate may have nascent bioactivity. The in vivo ratio orcomposition of BRs at different oxidation states may result in differentresponses. Noticeably distinctive phenotypes for the various BR dwarfs,defective in different biosynthetic steps, support this idea. Third, thebiosynthetic rate of each pathway toward production of the end productmay differ. In this case, the biosynthetic rate could be modulated bycontrolling the level of gene expression or the activity ofparticipating enzymes. Certain signals, requiring different rates of BRbiosynthesis, may induce one of the subpathways, which would then affectthe concentration of the intermediates in one pathway relative to theother.

[0230] Of the steps in BR biosynthesis in Madagascar periwinkle, the22α-hydroxylation reaction has been suggested to be the rate-limitingstep (Fujioka et al. (1995a) Biosci. Biotech. Biochem. 59:1543-1547). Inperiwinkle, the endogenous level of CT was as low as one-twentythousandth of CR; however, CT was almost 500 times more active than6-oxoCN in the rice-lamina inclination assay (Fujioka et al. (1995b)Biosci. Biotech. Biochem. 59:1973-1975). Based on these results, wepropose that the step encoded by DWF4 serves as the rate-limitingreaction and that once past this step, the intermediates are easilyconverted to the end product. Although biochemical studies on DWF4 needto be performed to ascertain whether it mediates the rate-limiting step,DWF4 seems to be greatly downregulated compared with CPD, the nextenzyme in the pathway; RT-PCR revealed that the DWF4 transcript is muchless abundant than the CPD transcript.

Example 11

[0231] A. Promoter and Overexpression Constructs

[0232] Two promoter constructs were used for the DWF4-promoter::GUS(D4G) analysis. For promoter fusions, polymerase chain reaction (PCR)products spanning 1.1 kb DNA upstream of the translation initiation sitewere amplified using primers D4XLNIT(5′-TAGGATCCAGCTAGTTTCTCTCTCTCTCT-3′) (SEQ ID NO:16) and a T7 primer(5′-TAATACGACTCACTATAGGG-3′) (SEQ ID NO:17). For template for PCR, a DWF4 genomic clone subcloned into pBluescript SK− vector (Stratagene, LaJolla, Calif.) was used as described herein. The PCR products wererestricted with SalI and BamHI, and ligated into the same restrictionsite of a promoterless GUS vector pBI 101; this 1.1 kb promoter:: GUSconstruct was named pD4GL. For the pD4GS construct, pD4GL was digestedwith HindIII, the small restriction fragment was removed, and theremaining vector with the partial promoter was self-ligated. Theconstructs were introduced into Agrobacterium strain GV3101 throughelectroporation.

[0233] For a DWF4 overexpression construct, PCR products were made byusing D40VERFA (5′-GAATTCTAGAATGTTCGAAACAGAGCATCATA-3′) (SEQ ID NO:18)and D4R2 (5′-CCGAACATCTTTGAGTGCTT-3′) (SEQ ID NO:10) primers andWassilewskija-2 (Ws-2) genomic DNA. The PCR products were cut with XbaIand HindIII, and inserted into the same restriction sites of genomicclone SCH25 containing a 2.5 kb HindIII fragment of the DWF4 DNAcorresponding to the 3′ half of the gene. The resulting recombinant DNAclone pD4CDS, containing the whole coding sequence from the translationinitiation site to 694 bp downstream of the stop codon, was cut withXbaI and transferred to an overexpression vector pART27 (Gleave (1992),Plant Molec. Bio. 20:1203-1207). The resulting binary construct wasnamed pOD4. This construct was introduced into Agrobacterium throughelectroporation.

[0234] B. Spray Transformation

[0235] Since it has been shown that Agrobacterium-mediatedtransformation can work by seed infection (Feldmann and Marks (1987)Molec. Gen. Genet. 208:1-9) or by simply dipping the host plants intoAgrobacterium culture, we decided to try spraying the Agrobacteriumdirectly onto the plants. In addition to spraying, the “floral dip”method was used as described (Clough and Bent (1998), infra). About 20Wassilewskija-2 (Ws-2) wild-type and dwf4-4 seeds were sprinkled on 10cm pots, and thinned to 5-6 plants per pot 10 days (wild type) and 20days (dwf4-4) after germination. When the primary inflorescences of thewild type reached 3-4 cm in height, they were decapitated to induceaxilary bolts. dwf4-4 plants were used without decapitation. For thepreparation of Agrobacterium, a single colony selected on 20 μg/mlkanamycin in Luria-Bertani (LB) medium (10 g bacto-tryptone, 5 gbacto-yeast extract, 10 g NaCl per liter, pH 7) was inoculated into 100ml liquid LB media, and grown for 3 days. One OD₆₀₀ unit equivalentcells were used to inoculate 100 ml LB media. The overnight grown cellswere collected by centrifugation, and resuspended with transformationmedia as described in Clough and Bent (1998), infra (5% sucrose and0.05% Silwet L-77, OD₆₀₀=1). The Agrobacterium suspension was sprayedonto plants on the third day after decapitation. To avoid physicalcontact with possibly hazardous Silwet vapor, protective glasses wereused and the spraying was done in a fume hood. To test the efficiency ofrepeated spraying, plants were sprayed every third day (3×). Sprayedplants were grown to maturity and seeds harvested. For seedsterilization 0.07 g seeds were surface sterilized by treating for 2 minin 70% ethanol, 15 min in bleach solution consisting of 5% Clorox and 1%SDS, followed by three rinses with sterile water. To plate the seeds 25ml of sterile top agar (0.15% agar) was added to the sterilized seedsand the seed mixture was poured onto Murashige and Skoog solid plate(100×15 mm, Murashige and Skoog salts, 5% sucrose, 0.08% agar, pH 6)supplemented with kanamycin or hygromycin at 60 μg/ml and 40 μg/ml,respectively. Twelve days after germination kanarnycin resistant weretransferred to single pots, and grown to maturity. T2 seeds werecollected from individual transformants (T1), and plated again on theselection media to determine segregation ratios for drug-resistantversus sensitive plants. Arabidopsis transformants were namedArabidopsis Overexpressor of DWF4 (AOD4) when harboring anoverexpression construct pOD4, and DWF4-promoter::GUS (D4G) fortransformants containing a GUS fusion gene. Homozygosity for thetransgene was determined when no sensitive T4 seedlings segregatedfrom >500 T3 individuals. Morphometric analysis of AOD4 lines and GUShistochemical analysis of D4GL plants was performed using plantshomozygous for the transgene.

[0236] For histochemical analysis of the D4GL plants, seeds were platedon M&S plates and grown in the dark and light. Seedlings were harvestedat the designated dates and stained overnight using a substrate mixture(0.1 M NaPO4, pH 7, 10 mM EDTA, 0.5 mM K₃Fe(CN)₆, 0.5 mM K₄Fe(CN)₆, 1 mMX-glucuronide, and 0.1% Triton X-100). Seedlings cleared with 90%ethanol were rehydrated before taking pictures using a Stemi SV11dissecting microscope (Zeiss, NY).

[0237] Transgenic tobacco plants (TOD4) harboring the pOD4 constructswere produced in the plant tissue culture laboratory at the Universityof Arizona. Protocols for the regeneration of transgenic plants fromlead discs of Nicotiana tabacum var Samsun will be provided on request.Fifteen independent transformants for both the control and OD4constructs were grown for seeds. Morphological analysis of the TOD4lines was performed using T2 plants in the course of growth for 4 monthsin the green house (30° C.). Methods for Arabidopsis growth and RNA gelblot analysis were previously described herein. Briefly, seeds of wildtype and the two AOD4 lines were germinated on M&S agar media. 10 daysafter germination, 20 seedlings confirmed to be resistant to kanamycinwere transferred to a single pot. Various morphological traits (Table 3)were measured. To determine the seed production, after 8 weeks fromgermination, plants were further dried for two weeks at roomtemperature. Seeds were harvested from an individual plant and weighed.To measure the seed size, seeds were magnified 3 times under thedissecting microscope, the width and the length of five seeds from eachplant were measured to the nearest tenth of mm.

[0238] C. DWF4 Transcription is Localized to Zones of Cell Division andElongation

[0239] To localize BR biosynthesis, RNA gel blot analysis with total RNAisolated from nine different tissues of three-week old plants wasperformed. The DWF4 transcript was barely detectable in shoot tips,roots, dark-grown seedlings, callus and axilary buds, but the levelswere below the detectable limit in the other tissues examined, includingstems, siliques, pedicels, and rosette leaves. For finer localization ofthe expression, the expression of the GUS reporter gene controlled bythe DWF4 promoter was examined.

[0240] Prior to performing DWF4-promoter::GUS gene fusion analysis, a1.1 kb fragment of DNA upstream of the DWF4 translation start site wastested to ensure that it contained all of the necessary sequenceelements for proper transcriptional control of DWF4. dwf4-4 plants weretransformed with a 4.8 kb construct consisting of a 1.1 kb promoterregion and 3.7 kb that contained the complete DWF4 coding sequence. ForAgrobacterium-mediated transformation of Arabidopsis plants, a “spraytransformation” protocol was employed rather than traditional methods.Spray transformation yielded a comparable number of transformantsrelative to the traditional “floral dip” (Clough and Bent (1998) PlantJ. 16:735-743) or “vacuum infiltration” methods (Bechtold et al. (1998)Methods Mol. Biol. 82:259-266). Interestingly, repeated sprayingresulted in an increased number of transformants. Transformantsharboring the DWF4 genomic DNA displayed a wild type phenotype,suggesting that the promoter segment contained the necessary informationfor proper expression of the gene.

[0241] For histochemical staining analyses of transgenic plantsharboring the DWF4-promoter::GUS (D4G) recombinant gene, two differentD4G constructs were made and tested. D4GL contained the 1.1 kb promoterfragment, whereas D4GS carried only a TATA-like promoter region (280bp). GUS staining in 20 independent transformants containing D4GS waseither not detected or inconsistent between transformants. However, the20 transgenic plants containing D4GL displayed a consistent GUS stainingpattern, suggesting that the 1.1 kb promoter is required for the propertranscriptional control of DWF4.

[0242] Analyses of GUS staining patterns in T2 plants homozygous forD4GL revealed that GUS activity was present in tissues with activelydividing or elongating cells. These include shoot apical meristems, leafprimordia, collet (the junction between hypocotyl and root), and roottips, including lateral root primordia, as shown in 6-day oldlight-grown seedlings. Interestingly, dark-grown seedlings displayed GUSactivity in cotyledons whereas the staining was not detectable in thecotyledons of light-grown seedlings. In adult plants, GUS activity wasdetected in floral primordia, carpels, and the basal end of thefilaments of unopened flowers, whereas GUS activity in sepals, petals,and mature pedicels was not detected. The shoot tips, bases of emergingbranches, and primordia of axilary inflorescences were GUS positive,whereas elongated internodes were negative. Embryos in the seeds of thefully elongated siliques were weakly positive for GUS staining,suggesting a role for BRs in embryo development. Leaf primordia, youngleaves, expanding leaf margins, and the base of petioles displayed GUSactivity, but old leaf blades were negative for GUS staining. Thetissues positive for GUS staining confirmed the expression patternexamined by northern analysis with the tissue-specific RNA.

[0243] Since DWF4 is proposed to be a key enzyme in the BR biosyntheticpathway, DWF4 transcription could be regulated by an end-productfeedback mechanism. To this end, D4GL was expressed in different geneticbackgrounds including two BR deficient mutants, dwf7-1 and dwf8-1, and aBR-enriched line, AOD4. GUS activity was increased in dwf7-1 and dwf8-1but decreased in AOD4 lines. DWF7 is a C-5 desaturase that acts in thesterol specific part of the pathway. D4GL activity in dwf7-1 was foundin the same tissues as wild type but. dwf8-1 is defective in a BRbiosynthetic step downstream of CPD. In dwf8-1, the intensity of theD4GL activity was noticeably stronger as compared to wild type but theexpression patterns were relatively diffuse. dwf8-1 was also found toexpress GUS at nascent sites as compared to wild type. In wild type,D4GL expression in the cotyledons of light-grown seedlings was notdetected, but dwf8-1 displayed considerable D4GL activity in thecotyledons. Also in contrast to wild type, GUS activity was detectedthroughout the hypocotyls of dwj8-1 light-grown seedlings, suggestingthat D4GL transcription is upregulated in dwf8-1 in a more generalmanner. Conversely, GUS activity was greatly reduced in AOD4-4 plants.Also, in AOD4 plants GUS activity in the root tip and collet wascompletely eliminated, whereas the shoot tip retained residual activity,suggesting that increased levels of BRs in AOD4-4 may have resulted inlower GUS activity. The down-regulation of GUS activity was similarlyfound if D4GL plants were exogenously supplied with 10⁻⁶ M24-epibrassinolide (epi-BL). Seedlings treated with epi-BL displayedgreatly reduced GUS activity in tissues normally stained in untreatedcontrol plants, suggesting that exogenously applied epi-BL effectivelydown-regulates D4GL activity. However, hypocotyls of D4GL plantssupplemented with 10⁻⁴ M GA₃, while longer than controls, did notdisplay an increase in GUS staining in shoots and roots. This suggeststhat GA₃ or GA₃-induced elongation did not affect D4GL transcription inthese tissues.

[0244] D. DWF4 Overexpression Results in Elongated Hypocotyls inArabidopsis and Tobacco Seedlings

[0245] A DWF4 overexpression construct (pOD4) was made by placing theDWF4 genomic DNA under the control of the CaMV 35S promoter. RNA gelblot analysis, with total RNA isolated from the transgenic linescontaining the overexpression construct, showed that DWF4 transcriptswere greatly increased in both Arabidopsis and tobacco, whereas thelevel was not readily detectable in either wild type or in dwf4-1plants. Similar to increased mRNA transcripts, the 80 independent AOD4transgenic plants had longer hypocotyls and inflorescences.

[0246] To compare the phenotypic effects resulting from the endogenousand exogenous addition of BRs, the length of roots and hypocotyls of 16seedlings of dwf4, wild-type controls, wild-type plants supplementedwith 10⁻⁶ M epi-BL, and two independent AOD4 lines, grown for 12 days inthe light or dark was measured. As described herein, dwf4-1 displayedgreatly reduced hypocotyl length both in the light and dark as comparedto wild type. Wild-type roots are shortened when grown in the dark, butdwf4-1 root length was not significantly reduced in the dark comparedwith the reduction in hypocotyl length. When epi-BL is added,light-grown wild type seedlings developed elongated hypocotyls, whereasroots were shorter than untreated control plants. These characteristicresponses of wild-type plants to epi-BL treatment were similar in twoindependent AOD4 lines. The hypocotyl length of light-grown AOD4seedlings was comparable to that of seedlings treated exogenously withepi-BL. However, dark-grown hypocotyls showed a dramatic increase inlength as compared to controls with or without epi-BL. Inhibition ofroot growth was also obvious in the AOD4 lines. Furthermore, theincreased hypocotyl length and reduced root length were consistentlyobserved in 15 independent transformants of tobacco (TOD4) harboring apOD4 construct. This result suggests that the Arabidopsis DWF4 enzymealso catalyzes BR biosynthesis in tobacco.

[0247] E. DWF4 Overexpression Results in Increased Plant Height, BiggerLeaves, and Increased Seed Production

[0248] As shown below in Table 3, the effects of DWF4 overexpression onplant growth were monitored during the course of development. The numberof rosette leaves at bolting was not significantly different betweenwild-type and AOD4 plants (Table 3). The inflorescence height of wildtype and two independent AOD4 lines were comparable 20 days aftergermination (DAG). Later, the AOD4 lines outgrow wild type.Surprisingly, AOD4 lines continue to grow beyond 35 DAG at the timewild-type plants ceased elongation. At maturity, the height of AOD4lines was 135% (AOD4-65) and 142% (AOD4-73) that of wild type,respectively. Similarly, TOD4 also displayed a 14% increase in plantheight as compared to the control. Interestingly, the increasedinflorescence length in AOD4 plants seemed to be at the cost of stemstiffness. During development, AOD4 plants tend to fall over earlierthan the Ws-2 wild type. In addition to plant height, comparison ofrosette leaf size between wild type and AOD4 indicates that leaves, bothrosette and cauline, are larger, especially in adult plants. TOD4 plantsalso possessed leaves that were larger, and had longer petioles relativeto the control. Furthermore, additional secondary branches were foundboth in Arabidopsis and tobacco overexpression lines. In AOD4 plants,this additional branching was associated with >2 times increased numberof siliques per plant, leading to a 33 and 59% increase in seedproduction (Table 3). The increased seed production in the AOD4 lineswas mainly due to the increased number of seeds per plant than increasein the seed size, because the size was not significantly increased(Table 3). In addition to the increased number of seeds, the length ofsilique as well as the length of an internode between the first siliquein a main inflorescence and the base of plant was increased (Table 3).

[0249]FIG. 8 shows that stem growth is increased more than 20% comparedto wild type in DWF4 overexpression lines and FIG. 9 shows that seedproduction is increased significantly over wild type in theDWF4-overexpressed lines. FIG. 12 depicts hypocotyl length and rootlength in light and dark. Further, the height of AOD4 lines was greaterthan wild type over the days examined. In addition, although wild typeplants ceased growth around five weeks after germination, AOD4 plantscontinued to grow up to seven weeks. TABLE 3 Morphological comparisonamong control and AOD4 lines. Percent increase over Ws-2 Ws-2 AOD4-65AOD4-73 AOD4-65 AOD4-73 Size Height in cm¹ 11.4 ± 1.8  15.4 ± 3.3  16.8± 3.8  135 147 Silique in mm 13.7 ± 2.0  18.4 ± 2.5  20.0 ± 3.2  134 145Seed length in mm 0.47 ± 0.04 0.51 ± 0.05 0.49 ± 0.03 108 105 Seed widthin mm 0.29 ± 0.03 0.32 ± 0.03  0.3 ± 0.03 110 102 Number Rosette 5.1 ±0.5 5.2 ± 0.4 5.7 ± 0.5 100 112 Branch² 9 ± 3 23.4 ± 5.7  21.6 ± 6.8 260 240 Siliques 106.7 ± 33.6  373.5 ± 107.5 344.2 ± 47.1  350 325 Seedproduction³ 164 ± 48  218 ± 33  261 ± 37  133 159 (mg/plant)

[0250] Thus, the DWF4 locus is defined by at least four mutant alleles.One of these is the result of a T-DNA insertion. Plant DNA flanking theinsertion site was cloned and used as a probe to isolate the entire DWF4gene. Sequence analysis revealed that DWF4 encodes a cytochrome P450monooxygenase with 43% identity to the putative Arabidopsis steroidhydroxylating enzyme CONSTITUTIVE PHOTOMORPHOGENESIS AND DWARFISM.Sequence analysis of two other mutant alleles revealed deletions or apremature stop codon, confirming that DWF4 had been cloned. Thissequence similarity suggests that DWF4 functions in specifichydroxylation steps during BR biosynthesis. The dwarf phenotype can berescued with exogenously supplied brassinolide. dwf4 mutants displayfeatures of light-regulatory mutants, but the dwarfed phenotype isentirely and specifically brassinosteroid dependent; no other hormonecan rescue dwf4 to a wild-type phenotype. Feeding studies utilizing BRintermediates showed that only 22α-hydroxylated BRs rescued the dwf4phenotype, confirming that DWF4 acts as a 22α-hydroxylase. In adultplants, strong GUS staining (indicative of dwf4 expression) was found inthe primordia of axilary inflorescences and secondary branches, and inyoung developing flowers. GUS expressing tissues correspond to thetissues sensitive to exogenously applied BRs leading to the hypothesisthat these tissues are putative brassinolide biosynthetic sites. Theinflorescence height of DWF4 overexpressing lines increased >35% inArabidopsis (AOD4) and 14% in tobacco (TOD4) as compared to controlplants at maturity. The total number of branches and siliquesincreased >2-fold in AOD4 plants, leading up to a 59% increase in seedproduction. The phenotypes of dwf4, D WF4, and AOD4 plants suggest thatthe degree of D WF4 transcription is associated with the degree of BReffects. In sum, it appears be possible to engineer agricultural plantswith increased biomass and seed yield.

1 30 1 6888 DNA Arabidopsis thaliana 1 atgtgggtat tatattgttg ggttcggtttgagctacaat ataaatttcg tgtttctggt 60 tattctgttc acatgatttg agtttggttctcaatttgga ttccaagata attaaatatt 120 aaaattcatt taaaatattt acaagtaattaattatcttt acattgtatt gttataacaa 180 aatatctatc tttggtatat gagaaaatatggagtttgga atttataata ataaaggaaa 240 taatcgattc catttggttg gattacacagttaagttttt gtgtttcttt tgttatatgt 300 atatgagtaa atcaaaaaga gtattgattgaagtgtaaac atatttcgtt atgaccccca 360 aaaaaaaaaa aaaaacaaac aaacaaaccccccccccgat atagtttttg gttctggatt 420 aggtttattt gatcataatt acatgcatcatttctttgat tactatgaag attttcttac 480 caattaaaat ttcgaattca tatctcttgattattaaatt aaatacgagt gtgaatatcc 540 gtttatcgat cactccaatc atgattatgattcttgtgct aatccagcaa attattaaca 600 agagtattga gaaaaaaccg aaaataagaaaagggaaaga gtagtgaccc atggagtatg 660 tgaataatta tcaaagagaa taagagatgacaaccaaaag gttgtggaat aatggtccct 720 gccagctttc tctcacaatc aatatcgaccctatttggat tttctggata ttcgttaaaa 780 tttgcgataa cgattgtgaa aaatattttatttgttagct gatctcaata ttatgttcca 840 ggtatttgca taatcttctg tttaaagcatattttgtctt tctttttgtt tcgtttctct 900 taactatata ttatcgcgga tatatgataacaatgatata tcacaaaaca attgtctggg 960 accattttga ataaactttt tctcaaacattacgggacac tggactcgac ccttaaaata 1020 cgattttaca gcgtcactag ttgagattactagcataaag cataaaggac ccgttcaagc 1080 tatttataca aagttacaaa ctgaatatagcttgaaatcc tttagaaaat tttggaatta 1140 ccggttgtta tgtaaatata gatttagtggtaaacaaata tgttaatcaa ttagtggtca 1200 acatatacat aattccttac agaaaaaacaaacttaagag aagttaacat atccatatat 1260 gggtatgcta tacctttcac gtatgctatactagagacta aagaatagtt atgtgatgtc 1320 gataaatgaa attcacacgc gtggtaataattatgggacc gtatgttacg atcactgcaa 1380 atatcattct tggttggtca acaataaaaacaaaaacaag aaaaaaagaa aacgattttt 1440 cttggattcc attcaatgat ctaaaatgcatagatctttt gggttacagt ttcgaagtcc 1500 tctacaagcg tgtaaccatc tgcaactattaaattgcttt ctttaatgca tctttaacat 1560 atttattgtt agttggaatt taataagagcgaacttgtaa cattacaata tttatattag 1620 atactagtat gtgattattc caaatacatactttggatgt ttaaacttaa tcttgtttct 1680 tcctacggta taaatattaa tcatcgaggtaaaaaaagtt ttgtcttatt ttcgcgatgc 1740 atgaaggata aacctaatga ctttaattttttgaaaatgt aaccctttta ctcatagatt 1800 aattaccgta tgtttttgtt gccataatgacagcctctac aactgtgata gtcaattttt 1860 tctgcaaata ttaaattagg aattcaatgctactatcaat agaagaaaca gctgagtatt 1920 acattttaat ttaaagacaa aatttttgaaaaatgttata atttctaaca atattattaa 1980 aatatgatgc ctataatgta tttcctatgttcttaaaata ttttttttta tatttagtta 2040 taaatacatt atgaaccaat aatagttggtgaattcaaat atctccatta atattttttg 2100 aaatctacaa attattaata tttagtcaataacaatgcat agaaagttcc aaaaaaaatt 2160 ttgttaacag aaacttccaa attttttttttttatggaac aagaaataac agatagaaaa 2220 ctattttgtt gtggaatgga agtagtaatatacattaagc aaattttaaa aaattatata 2280 agcctatacg cgctcaaagt atgttatctagtaggtgtaa ttaataatgc atggtgcgat 2340 tcagaattgg gacaacaatg aaaacggaattaaaatatta actttaaaat aaataaaaat 2400 ttgagtaaat gtgttttctg actattgaggggcaaaaaaa agacaatgcc aaaagtctac 2460 gggtttgact gtccagttcg gtaataatctaataactctg tctttgaccg cacgctcgtg 2520 taggggtcct tctgacattt tcactgttctacccctactc gtgagcccac ccttttccca 2580 tatcctaagg gtaattttgg aaatcccaatttaaaccgat tgagaccgta ccggacttcc 2640 tgggattctg ctggagcatt tatcaaaaattattagcacg aatgggttta ttaatttaaa 2700 aactcacaac ttgatcagat aaaatttcataaacactttt acgatggatt cgtacgatct 2760 atctaatgac tttttttttt ctaccacggtggatgaaagt tatagtacta ttagccagag 2820 acaattgatt atagatatat ccattaatccatgatattta tgatataaat agctgttaaa 2880 ctatttcagc atcgcagctt tctgcaacttttgtttttaa tttaagagtt taataaataa 2940 aagtattaaa aggagcataa cgaggcaacaaaagtaatga acacggagaa acaaaagcca 3000 tgaagctcat tggttagttt aagcttaataagaagatttt attaaatttt aatgacgatg 3060 ataacaatta tattttctga cttctttaaaaccccctctt acaaacagaa gctccctttt 3120 tcagtagaag tccgattccc aatcttaaagacaaagccat tagaaagaga aagtgagtga 3180 gagagagaga gaaactagct ccatgttcgaaacagagcat catactctct tacctcttct 3240 tcttctccca tcgcttttgt ctcttcttctcttcttgatt ctcttgaaga gaagaaatag 3300 aaaaaccaga ttcaatctac ctccgggtaaatccggttgg ccatttcttg gtgaaaccat 3360 cggttatctt aaaccgtaca ccgccacaacactcggtgac ttcatgcaac aacatgtctc 3420 caagtaaaca acaacatctt ccaaaaactcaaaaaaataa atcctctgtt tttgaaattt 3480 gactaatgtt gtttatttta caggtatggtaagatatata gatcgaactt gtttggagaa 3540 ccaacgatcg tatcagctga tgctggacttaatagattca tattacaaaa cgaaggaagg 3600 ctctttgaat gtagttatcc tagaagtataggtgggattc ttgggaaatg gtcgatgctt 3660 gttcttgttg gtgacatgca tagagatatgagaagtatct cgcttaactt cttaagtcac 3720 gcacgtctta gaactattct acttaaagatgttgagagac atactttgtt tgttcttgat 3780 tcttggcaac aaaactctat tttctctgctcaagacgagg ccaaaaaggt ttttattttt 3840 atcttttatt ttgctaaatt tttttgtttatgaatcttta gagtttctaa cttttttttt 3900 tttaattgaa cagtttacgt ttaatctaatggcgaagcat ataatgagta tggatcctgg 3960 agaagaagaa acagagcaat taaagaaagagtatgtaact ttcatgaaag gagttgtctc 4020 tgctcctcta aatctaccag gaactgcttatcataaagct cttcaggtac atttattttt 4080 ttttgctgta aagtcacaaa ctctcattataggtttttaa ttttatttta tgtgttaaat 4140 aaaatatcta aaatggttgt gtagtcacgagcaacgatat tgaagttcat tgagaggaaa 4200 atggaagaga gaaaattgga tatcaaggaagaagatcaag aagaagaaga agtgaaaaca 4260 gaggatgaag cagagatgag taagagtgatcatgttagga aacaaagaac agacgatgat 4320 cttttgggat gggttttgaa acattcgaatttatcgacgg agcaaattct cgatctcatt 4380 cttagtttgt tatttgccgg acatgagacttcttctgtag ccattgctct cgctatcttc 4440 ttcttgcaag cttgccctaa agccgttgaagagcttaggg taagataatt ataacagcac 4500 aagttaatta ctaccaaatt gttacgtattatataagtta ttatagaatt attctattag 4560 aatatacgat gaaaaaagta tgtatatttaattgtcacta attttatgtt tattgattta 4620 tacttttgaa ggaagagcat cttgagatcgcgagggccaa gaaggaacta ggagagtcag 4680 aattaaattg ggatgattac aagaaaatggactttactca atgtgtatgt tactatcatt 4740 ctcattattt attctatgtt catatgatttatgatgaaac caaaattatt gatttttttt 4800 ttggtgtgtg tgaaggttat aaatgaaactcttcgattgg gaaatgtagt taggtttttg 4860 catcgcaaag cactcaaaga tgttcggtacaaaggtaaaa ctttacgtac aaaattttta 4920 aataatgaaa tccggaatat tgaaatcttattggatgaaa aatattaaaa taatttacat 4980 ttcttaatgt tggaaaaaag gatacgatatccctagtggg tggaaagtgt taccggtgat 5040 ctcagccgta catttggata attctcgttatgaccaacct aatctcttta atccttggag 5100 atggcaacag gtaaataaaa agtttctctcgttaactatc gaaaattagt gtatagtttt 5160 ttcatctatt gcatgaatag atacgtcctacgtgatttac ctatctatag atactatacg 5220 agaactatta atctggcaaa aactttttattattattatc tttcaagtta gatcttaaca 5280 cgtcatggat cattgatcac atgaaagcatataaattaaa aataagagag agaaagagac 5340 gtgttggtgt aagtgtacgt gaagacaattaattagtagg atggtatgtc tttaatgacg 5400 taggagctgc ctaaatattc ttataatcgtgaccgttgat ttattattag tcacggcttt 5460 gatacaattt aagatttgac ggacgatggtaccacggctt tgacggatct cacacgcccg 5520 atgacttgta cgtgcgttag attctgccacgttgactggt tttaatactt agatttataa 5580 ctctattaat tataacaact atcaaatcggcgaattagag aaatatacta tatagtatta 5640 ttatgattat tatgagataa tactttatgaaataagataa taatggtagt catgatgtta 5700 tagtgagtgg ggaaggtaag aggtggtgagagatgattaa tgaccccacg tggtgtggtg 5760 ccaacaagca cgtgttcttc ttccttttttcttcccaact tctttttttg ggggtttatt 5820 gtgatttata aaatcggttt gtcgtttttttttgtgacga gcagcaaaac aacggagcgt 5880 catcgtcagg aagtggtagt ttttcgacgtggggaaacaa ctacatgccg tttggaggag 5940 ggccaaggct atgtgctggt tcagagctagccaagttaga aatggcagtg tttattcatc 6000 atctagttct taaattcaat tgggaattagcagaagatga tcaaccattt gcttttcctt 6060 ttgttgattt tcctaacggt ttgcctattagggtttctcg tattctgtaa aaaaaaaaaa 6120 agatgaaagt atttttattc tcttcttttttttttgataa ttttaaatca ttttttttgc 6180 ccaatgatat ataaaaattt ggataaataatattattgga tattcgtttt ttagttcggg 6240 tttgagaaaa gggtttcgac tttcgaaagtggacgatgta tatagattgg gagctaggtt 6300 gagtctttgg acatttgtat tggatgttgttgattattag tgtcgacact attaaacctt 6360 aaatgggctt tctataaggc ccaattatattacgattata acaaagtgac aacttttact 6420 tcgtttttga tccgaagcaa taacaaattgtcaaatacca aacacaagaa ttatgtaaac 6480 actcgtgtgt gtctagtggg aaatcattgggctggagact gaacatcaga acacaagaaa 6540 cctgtcaatt atggatacac ctcctatgacggtttccaaa ctttatcttg attcttatcg 6600 tgttacattg acacaaagag ttaggtgtcaaaaggactaa atgaataaca atagctctca 6660 ggataagaag gttcataaaa tggtttctttattttgagaa gaaagagaga ggagctttta 6720 ctgtttcttg ggtcctattc ctttaaatgagagggtttcg tttttacttc ttctatctca 6780 tcatctttag gatcctcttc tagacgagtaaagtaatcct cgttaccaag caatggtctc 6840 atcttttgaa gacaggtctt ttccaagtcctagttcaggc caaagctt 6888 2 513 PRT Arabidopsis thaliana 2 Met Phe GluThr Glu His His Thr Leu Leu Pro Leu Leu Leu Leu Pro 1 5 10 15 Ser LeuLeu Ser Leu Leu Leu Phe Leu Ile Leu Leu Lys Arg Arg Asn 20 25 30 Arg LysThr Arg Phe Asn Leu Pro Pro Gly Lys Ser Gly Trp Pro Phe 35 40 45 Leu GlyGlu Thr Ile Gly Tyr Leu Lys Pro Tyr Thr Ala Thr Thr Leu 50 55 60 Gly AspPhe Met Gln Gln His Val Ser Lys Tyr Gly Lys Ile Tyr Arg 65 70 75 80 SerAsn Leu Phe Gly Glu Pro Thr Ile Val Ser Ala Asp Ala Gly Leu 85 90 95 AsnArg Phe Ile Leu Gln Asn Glu Gly Arg Leu Phe Glu Cys Ser Tyr 100 105 110Pro Arg Ser Ile Gly Gly Ile Leu Gly Lys Trp Ser Met Leu Val Leu 115 120125 Val Gly Asp Met His Arg Asp Met Arg Ser Ile Ser Leu Asn Phe Leu 130135 140 Ser His Ala Arg Leu Arg Thr Ile Leu Leu Lys Asp Val Glu Arg His145 150 155 160 Thr Leu Phe Val Leu Asp Ser Trp Gln Gln Asn Ser Ile PheSer Ala 165 170 175 Gln Asp Glu Ala Lys Lys Phe Thr Phe Asn Leu Met AlaLys His Ile 180 185 190 Met Ser Met Asp Pro Gly Glu Glu Glu Thr Glu GlnLeu Lys Lys Glu 195 200 205 Tyr Val Thr Phe Met Lys Gly Val Val Ser AlaPro Leu Asn Leu Pro 210 215 220 Gly Thr Ala Tyr His Lys Ala Leu Gln SerArg Ala Thr Ile Leu Lys 225 230 235 240 Phe Ile Glu Arg Lys Met Glu GluArg Lys Leu Asp Ile Lys Glu Glu 245 250 255 Asp Gln Glu Glu Glu Glu ValLys Thr Glu Asp Glu Ala Glu Met Ser 260 265 270 Lys Ser Asp His Val ArgLys Gln Arg Thr Asp Asp Asp Leu Leu Gly 275 280 285 Trp Val Leu Lys HisSer Asn Leu Ser Thr Glu Gln Ile Leu Asp Leu 290 295 300 Ile Leu Ser LeuLeu Phe Ala Gly His Glu Thr Ser Ser Val Ala Ile 305 310 315 320 Ala LeuAla Ile Phe Phe Leu Gln Ala Cys Pro Lys Ala Val Glu Glu 325 330 335 LeuArg Glu Glu His Leu Glu Ile Ala Arg Ala Lys Lys Glu Leu Gly 340 345 350Glu Ser Glu Leu Asn Trp Asp Asp Tyr Lys Lys Met Asp Phe Thr Gln 355 360365 Cys Val Ile Asn Glu Thr Leu Arg Leu Gly Asn Val Val Arg Phe Leu 370375 380 His Arg Lys Ala Leu Lys Asp Val Arg Tyr Lys Gly Tyr Asp Ile Pro385 390 395 400 Ser Gly Trp Lys Val Leu Pro Val Ile Ser Ala Val His LeuAsp Asn 405 410 415 Ser Arg Tyr Asp Gln Pro Asn Leu Phe Asn Pro Trp ArgTrp Gln Gln 420 425 430 Gln Asn Asn Gly Ala Ser Ser Ser Gly Ser Gly SerPhe Ser Thr Trp 435 440 445 Gly Asn Asn Tyr Met Pro Phe Gly Gly Gly ProArg Leu Cys Ala Gly 450 455 460 Ser Glu Leu Ala Lys Leu Glu Met Ala ValPhe Ile His His Leu Val 465 470 475 480 Leu Lys Phe Asn Trp Glu Leu AlaGlu Asp Asp Gln Pro Phe Ala Phe 485 490 495 Pro Phe Val Asp Phe Pro AsnGly Leu Pro Ile Arg Val Ser Arg Ile 500 505 510 Leu 3 24 DNA ArtificialSequence Primer D4OVERF 3 atgttcgaaa cagagcatca tact 24 4 21 DNAArtificial Sequence Primer D4PRM 4 cctcgatcaa agagagagag a 21 5 29 DNAArtificial Sequence Primer D4RTF 5 ttcttggtga aaccatcggt tatcttaaa 29 626 DNA Artificial Sequence Primer D4RTR 6 tatgataagc agttcctggt agattt26 7 21 DNA Artificial Sequence Primer D4F1 7 cgaggcaaca aaagtaatga a 218 21 DNA Artificial Sequence Primer D4R1 8 gttagaaact ctaaagattc a 21 923 DNA Artificial Sequence Primer D4F2 9 gattcttggc aacaaaactc tat 23 1020 DNA Artificial Sequence Primer D4R2 10 ccgaacatct ttgagtgctt 20 11 26DNA Artificial Sequence Primer D4F3 11 gtgtgaaggt tataaatgaa actctt 2612 24 DNA Artificial Sequence Primer D4R3 12 ggtttaatag tgtcgacact aata24 13 22 DNA Artificial Sequence Primer D4F4 13 ccgatgactt gtacgtgcgt ta22 14 24 DNA Artificial Sequence Primer D4F5 14 gcgaagcata taatgagtatggat 24 15 26 DNA Artificial Sequence Primer D4R5 15 gttggtcataacgagaatta tccaaa 26 16 29 DNA Artificial Sequence Primer D4XLINIT 16taggatccag ctagtttctc tctctctct 29 17 20 DNA Artificial Sequence PrimerT7 17 taatacgact cactataggg 20 18 32 DNA Artificial Sequence PrimerD4OVERFA 18 gaattctaga atgttcgaaa cagagcatca ta 32 19 472 PRTArabidopsis thaliana 19 Met Ala Phe Thr Ala Phe Leu Leu Leu Leu Ser SerIle Ala Ala Gly 1 5 10 15 Phe Leu Leu Leu Leu Arg Arg Thr Arg Tyr ArgArg Met Gly Leu Pro 20 25 30 Pro Gly Ser Leu Gly Leu Pro Leu Ile Gly GluThr Phe Gln Leu Ile 35 40 45 Gly Ala Tyr Lys Thr Glu Asn Pro Glu Pro PheIle Asp Glu Arg Val 50 55 60 Ala Arg Tyr Gly Ser Val Phe Met Thr His LeuPhe Gly Glu Pro Thr 65 70 75 80 Ile Phe Ser Ala Asp Pro Glu Thr Asn ArgPhe Val Leu Gln Asn Glu 85 90 95 Gly Lys Leu Phe Glu Cys Ser Tyr Pro AlaSer Ile Cys Asn Leu Leu 100 105 110 Gly Lys His Ser Leu Leu Leu Met LysGly Ser Leu His Lys Arg Met 115 120 125 His Ser Leu Thr Met Ser Phe AlaAsn Ser Ser Ile Ile Lys Asp His 130 135 140 Leu Met Leu Asp Ile Asp ArgLeu Val Arg Phe Asn Leu Asp Ser Trp 145 150 155 160 Ser Ser Arg Val LeuLeu Met Glu Glu Ala Lys Lys Ile Thr Phe Glu 165 170 175 Leu Thr Val LysGln Leu Met Ser Phe Asp Pro Gly Glu Trp Ser Glu 180 185 190 Ser Leu ArgLys Glu Tyr Leu Leu Val Ile Glu Gly Phe Phe Ser Leu 195 200 205 Pro LeuPro Leu Phe Ser Thr Thr Tyr Arg Lys Ala Ile Gln Ala Arg 210 215 220 ArgLys Val Ala Glu Ala Leu Thr Val Val Val Met Lys Arg Arg Glu 225 230 235240 Glu Glu Glu Glu Gly Ala Glu Arg Lys Lys Asp Met Leu Ala Ala Leu 245250 255 Leu Ala Ala Asp Asp Gly Phe Ser Asp Glu Glu Ile Val Asp Phe Leu260 265 270 Val Ala Leu Leu Val Ala Gly Tyr Glu Thr Thr Ser Thr Ile MetThr 275 280 285 Leu Ala Val Lys Phe Leu Thr Glu Thr Pro Leu Ala Leu AlaGln Leu 290 295 300 Lys Glu Glu His Glu Lys Ile Arg Ala Met Lys Ser AspSer Tyr Ser 305 310 315 320 Leu Glu Trp Ser Asp Tyr Lys Ser Met Pro PheThr Gln Cys Val Val 325 330 335 Asn Glu Thr Leu Arg Val Ala Asn Ile IleGly Gly Val Phe Arg Arg 340 345 350 Ala Met Thr Asp Val Glu Ile Lys GlyTyr Lys Ile Pro Lys Gly Trp 355 360 365 Lys Val Phe Ser Ser Phe Arg AlaVal His Leu Asp Pro Asn His Phe 370 375 380 Lys Asp Ala Arg Thr Phe AsnPro Trp Arg Trp Gln Ser Asn Ser Val 385 390 395 400 Thr Thr Gly Pro SerAsn Val Phe Thr Pro Phe Gly Gly Gly Pro Arg 405 410 415 Leu Cys Pro GlyTyr Glu Leu Ala Arg Val Ala Leu Ser Val Phe Leu 420 425 430 His Arg LeuVal Thr Gly Phe Ser Trp Val Pro Ala Glu Gln Asp Lys 435 440 445 Leu ValPhe Phe Pro Thr Thr Arg Thr Gln Lys Arg Tyr Pro Ile Phe 450 455 460 ValLys Arg Arg Asp Phe Ala Thr 465 470 20 464 PRT Lycopersicon esculentum20 Met Ala Phe Phe Leu Ile Phe Leu Ser Ser Phe Phe Gly Leu Cys Ile 1 510 15 Phe Cys Thr Ala Leu Leu Arg Trp Asn Gln Val Lys Tyr Asn Gln Lys 2025 30 Asn Leu Pro Pro Gly Thr Met Gly Trp Pro Leu Phe Gly Glu Thr Thr 3540 45 Glu Phe Leu Lys Leu Gly Pro Ser Phe Met Lys Asn Gln Arg Ala Arg 5055 60 Tyr Gly Ser Phe Phe Lys Ser His Ile Leu Gly Cys Pro Thr Ile Val 6570 75 80 Ser Met Asp Ser Glu Leu Asn Arg Tyr Ile Leu Val Asn Glu Ala Lys85 90 95 Gly Leu Val Pro Gly Tyr Pro Gln Ser Met Ile Asp Ile Leu Gly Lys100 105 110 Cys Asn Ile Ala Ala Val Asn Gly Ser Ala His Lys Tyr Met ArgGly 115 120 125 Ala Leu Leu Ser Leu Ile Ser Pro Thr Met Ile Arg Asp GlnLeu Leu 130 135 140 Pro Lys Ile Asp Glu Phe Met Arg Ser His Leu Thr AsnTrp Asp Asn 145 150 155 160 Lys Val Ile Asp Ile Gln Glu Lys Thr Asn LysMet Ala Phe Leu Ser 165 170 175 Ser Leu Lys Gln Ile Ala Gly Ile Glu SerThr Ser Leu Ala Gln Glu 180 185 190 Phe Met Ser Glu Phe Phe Asn Leu ValLeu Gly Thr Leu Ser Leu Pro 195 200 205 Ile Asn Leu Pro Asn Thr Asn TyrHis Arg Gly Phe Gln Ala Arg Lys 210 215 220 Ile Ile Val Asn Leu Leu ArgThr Leu Ile Glu Glu Arg Arg Ala Ser 225 230 235 240 Lys Glu Ile Gln HisAsp Met Leu Gly Tyr Leu Met Asn Glu Glu Ala 245 250 255 Thr Arg Phe LysLeu Thr Asp Asp Glu Met Ile Asp Leu Ile Ile Thr 260 265 270 Ile Leu TyrSer Gly Tyr Glu Thr Val Ser Thr Thr Ser Met Met Ala 275 280 285 Val LysTyr Leu His Asp His Pro Lys Val Leu Glu Glu Leu Arg Lys 290 295 300 GluHis Met Ala Ile Arg Glu Lys Lys Lys Pro Glu Asp Pro Ile Asp 305 310 315320 Tyr Asn Asp Tyr Arg Ser Met Arg Phe Thr Arg Ala Val Ile Leu Glu 325330 335 Thr Ser Arg Leu Ala Thr Ile Val Asn Gly Val Leu Arg Lys Thr Thr340 345 350 Gln Asp Met Glu Ile Asn Gly Tyr Ile Ile Pro Lys Gly Trp ArgIle 355 360 365 Tyr Val Tyr Thr Arg Glu Leu Asn Tyr Asp Pro Arg Leu TyrPro Asp 370 375 380 Pro Tyr Ser Phe Asn Pro Trp Arg Trp Met Asp Lys SerLeu Glu His 385 390 395 400 Gln Asn Ser Phe Leu Val Phe Gly Gly Gly ThrArg Gln Cys Pro Gly 405 410 415 Lys Glu Leu Gly Val Ala Glu Ile Ser ThrPhe Leu His Tyr Phe Val 420 425 430 Thr Lys Tyr Arg Trp Glu Glu Ile GlyGly Asp Lys Leu Met Lys Phe 435 440 445 Pro Arg Val Glu Ala Pro Asn GlyLeu Arg Ile Arg Val Ser Ala His 450 455 460 21 444 PRT Synechocystis sp.21 Met Ile Thr Ser Pro Thr Asn Leu Asn Ser Leu Pro Ile Pro Pro Gly 1 510 15 Asp Phe Gly Leu Pro Trp Leu Gly Glu Thr Leu Asn Phe Leu Asn Asp 2025 30 Gly Asp Phe Gly Lys Lys Arg Gln Gln Gln Phe Gly Pro Ile Phe Lys 3540 45 Thr Arg Leu Phe Gly Lys Asn Val Ile Phe Ile Ser Gly Ala Leu Ala 5055 60 Asn Arg Phe Leu Phe Thr Lys Glu Gln Glu Thr Phe Gln Ala Thr Trp 6570 75 80 Pro Leu Ser Thr Arg Ile Leu Leu Gly Pro Asn Ala Leu Ala Thr Gln85 90 95 Met Gly Glu Ile His Arg Ser Arg Arg Lys Ile Leu Tyr Gln Ala Phe100 105 110 Leu Pro Arg Thr Leu Asp Ser Tyr Leu Pro Lys Met Asp Gly IleVal 115 120 125 Gln Gly Tyr Leu Glu Gln Trp Gly Lys Ala Asn Glu Val IleTrp Tyr 130 135 140 Pro Gln Leu Arg Arg Met Thr Phe Asp Val Ala Ala ThrLeu Phe Met 145 150 155 160 Gly Glu Lys Val Ser Gln Asn Pro Gln Leu PhePro Trp Phe Glu Thr 165 170 175 Tyr Ile Gln Gly Leu Phe Ser Leu Pro IlePro Leu Pro Asn Thr Leu 180 185 190 Phe Gly Lys Ser Gln Arg Ala Arg AlaLeu Leu Leu Ala Glu Leu Glu 195 200 205 Lys Ile Ile Lys Ala Arg Gln GlnGln Pro Pro Ser Glu Glu Asp Ala 210 215 220 Leu Gly Ile Leu Leu Ala AlaArg Asp Asp Asn Asn Gln Pro Leu Ser 225 230 235 240 Leu Pro Glu Leu LysAsp Gln Ile Leu Leu Leu Leu Phe Ala Gly His 245 250 255 Glu Thr Leu ThrSer Ala Leu Ser Ser Phe Cys Leu Leu Leu Gly Gln 260 265 270 His Ser AspIle Arg Glu Arg Val Arg Gln Glu Gln Asn Lys Leu Gln 275 280 285 Leu SerGln Glu Leu Thr Ala Glu Thr Leu Lys Lys Met Pro Tyr Leu 290 295 300 AspGln Val Leu Gln Glu Val Leu Arg Leu Ile Pro Pro Val Gly Gly 305 310 315320 Gly Phe Arg Glu Leu Ile Gln Asp Cys Gln Phe Gln Gly Phe His Phe 325330 335 Pro Lys Gly Trp Leu Val Ser Tyr Gln Ile Ser Gln Thr His Ala Asp340 345 350 Pro Asp Leu Tyr Pro Asp Pro Glu Lys Phe Asp Pro Glu Arg PheThr 355 360 365 Pro Asp Gly Ser Ala Thr His Asn Pro Pro Phe Ala His ValPro Phe 370 375 380 Gly Gly Gly Leu Arg Glu Cys Leu Gly Lys Glu Phe AlaArg Leu Glu 385 390 395 400 Met Lys Leu Phe Ala Thr Arg Leu Ile Gln GlnPhe Asp Trp Thr Leu 405 410 415 Leu Pro Gly Gln Asn Leu Glu Leu Val ValThr Pro Ser Pro Arg Pro 420 425 430 Lys Asp Asn Leu Arg Val Lys Leu HisSer Leu Met 435 440 22 519 PRT Zea mays 22 Met Leu Gly Val Gly Met AlaAla Ala Val Leu Leu Gly Ala Val Ala 1 5 10 15 Leu Leu Leu Ala Asp AlaAla Ala Arg Arg Ala His Trp Trp Tyr Arg 20 25 30 Glu Ala Ala Glu Ala ValLeu Val Gly Ala Val Ala Leu Val Val Val 35 40 45 Asp Ala Ala Ala Arg ArgAla His Gly Trp Tyr Arg Glu Ala Ala Leu 50 55 60 Gly Ala Ala Arg Arg AlaArg Leu Pro Pro Gly Glu Met Gly Trp Pro 65 70 75 80 Leu Val Gly Gly MetTrp Ala Phe Leu Arg Ala Phe Lys Ser Gly Lys 85 90 95 Pro Asp Ala Phe IleAla Ser Phe Val Arg Arg Phe Gly Arg Thr Gly 100 105 110 Val Tyr Arg SerPhe Met Phe Ser Ser Pro Thr Val Leu Val Thr Thr 115 120 125 Ala Glu GlyCys Lys Gln Val Leu Met Asp Asp Asp Ala Phe Val Thr 130 135 140 Gly TrpPro Lys Ala Thr Val Ala Leu Val Gly Pro Arg Ser Phe Val 145 150 155 160Ala Met Pro Tyr Asp Glu His Arg Arg Ile Arg Lys Leu Thr Ala Ala 165 170175 Pro Ile Asn Gly Phe Asp Ala Leu Thr Gly Tyr Leu Pro Phe Ile Asp 180185 190 Arg Thr Val Thr Ser Ser Leu Arg Ala Trp Ala Asp His Gly Gly Ser195 200 205 Val Glu Phe Leu Thr Glu Leu Arg Arg Met Thr Phe Lys Ile IleVal 210 215 220 Gln Ile Phe Leu Gly Gly Ala Asp Gln Ala Thr Thr Arg AlaLeu Glu 225 230 235 240 Arg Ser Tyr Thr Glu Leu Asn Tyr Gly Met Arg AlaMet Ala Ile Asn 245 250 255 Leu Pro Gly Phe Ala Tyr Arg Gly Ala Leu ArgAla Arg Arg Arg Leu 260 265 270 Val Ala Val Leu Gln Gly Val Leu Asp GluArg Arg Ala Ala Arg Ala 275 280 285 Lys Gly Val Ser Gly Gly Gly Val AspMet Met Asp Arg Leu Ile Glu 290 295 300 Ala Gln Asp Glu Arg Gly Arg HisLeu Asp Asp Asp Glu Ile Ile Asp 305 310 315 320 Val Leu Val Met Tyr LeuAsn Ala Gly His Glu Ser Ser Gly His Ile 325 330 335 Thr Met Trp Ala ThrVal Phe Leu Gln Glu Asn Pro Asp Met Phe Ala 340 345 350 Arg Ala Lys AlaGlu Gln Glu Ala Ile Met Arg Ser Ile Pro Ser Ser 355 360 365 Gln Arg GlyLeu Thr Leu Arg Asp Phe Arg Lys Met Glu Tyr Leu Ser 370 375 380 Gln ValIle Asp Glu Thr Leu Arg Leu Val Asn Ile Ser Phe Val Ser 385 390 395 400Phe Arg Gln Ala Thr Arg Asp Val Phe Val Asn Gly Tyr Leu Ile Pro 405 410415 Lys Gly Trp Lys Val Gln Leu Trp Tyr Arg Ser Val His Met Asp Pro 420425 430 Gln Val Tyr Pro Asp Pro Thr Lys Phe Asp Pro Ser Arg Trp Glu Gly435 440 445 His Ser Pro Arg Ala Gly Thr Phe Leu Ala Phe Gly Leu Gly AlaArg 450 455 460 Leu Cys Pro Gly Asn Asp Leu Ala Lys Leu Glu Ile Ser ValPhe Leu 465 470 475 480 His His Phe Leu Leu Gly Tyr Lys Leu Ala Arg ThrAsn Pro Arg Cys 485 490 495 Arg Val Arg Tyr Leu Pro His Pro Arg Pro ValAsp Asn Cys Leu Ala 500 505 510 Lys Ile Thr Arg Val Gly Ser 515 23 492PRT Danio rerio 23 Met Gly Leu Tyr Thr Leu Met Val Thr Phe Leu Cys ThrIle Val Leu 1 5 10 15 Pro Val Leu Leu Phe Leu Ala Ala Val Lys Leu TrpGlu Met Leu Met 20 25 30 Ile Arg Arg Val Asp Pro Asn Cys Arg Ser Pro LeuPro Pro Gly Thr 35 40 45 Met Gly Leu Pro Phe Ile Gly Glu Thr Leu Gln LeuIle Leu Gln Arg 50 55 60 Arg Lys Phe Leu Arg Met Lys Arg Gln Lys Tyr GlyCys Ile Tyr Lys 65 70 75 80 Thr His Leu Phe Gly Asn Pro Thr Val Arg ValMet Gly Ala Asp Asn 85 90 95 Val Arg Gln Ile Leu Leu Gly Glu His Lys LeuVal Ser Val Gln Trp 100 105 110 Pro Ala Ser Val Arg Thr Ile Leu Gly SerAsp Thr Leu Ser Asn Val 115 120 125 His Gly Val Gln His Lys Asn Lys LysLys Ala Ile Met Arg Ala Phe 130 135 140 Ser Arg Asp Ala Leu Glu His TyrIle Pro Val Ile Gln Gln Glu Val 145 150 155 160 Lys Ser Ala Ile Gln GluTrp Leu Gln Lys Asp Ser Cys Val Leu Val 165 170 175 Tyr Pro Glu Met LysLys Leu Met Phe Arg Ile Ala Met Arg Ile Leu 180 185 190 Leu Gly Phe GluPro Glu Gln Ile Lys Thr Asp Glu Gln Glu Leu Val 195 200 205 Glu Ala PheGlu Glu Met Ile Lys Asn Leu Phe Ser Leu Pro Ile Asp 210 215 220 Val ProPhe Ser Gly Leu Tyr Arg Gly Leu Arg Ala Arg Asn Phe Ile 225 230 235 240His Ser Lys Ile Glu Glu Asn Ile Arg Lys Lys Ile Gln Asp Asp Asp 245 250255 Asn Glu Asn Glu Gln Lys Tyr Lys Asp Ala Leu Gln Leu Leu Ile Glu 260265 270 Asn Ser Arg Arg Ser Asp Glu Pro Phe Ser Leu Gln Ala Met Lys Glu275 280 285 Ala Ala Thr Glu Leu Leu Phe Gly Gly His Glu Thr Thr Ala SerThr 290 295 300 Ala Thr Ser Leu Val Met Phe Leu Gly Leu Asn Thr Glu ValVal Gln 305 310 315 320 Lys Val Arg Glu Glu Val Gln Glu Lys Val Glu MetGly Met Tyr Thr 325 330 335 Pro Gly Lys Gly Leu Ser Met Glu Leu Leu AspGln Leu Lys Tyr Thr 340 345 350 Gly Cys Val Ile Lys Glu Thr Leu Arg IleAsn Pro Pro Val Pro Gly 355 360 365 Gly Phe Arg Val Ala Leu Lys Thr PheGlu Leu Asn Gly Tyr Gln Ile 370 375 380 Pro Lys Gly Trp Asn Val Ile TyrSer Ile Cys Asp Thr His Asp Val 385 390 395 400 Ala Asp Val Phe Pro AsnLys Glu Glu Phe Gln Pro Glu Arg Phe Met 405 410 415 Ser Lys Gly Leu GluAsp Gly Ser Arg Phe Asn Tyr Ile Pro Phe Gly 420 425 430 Gly Gly Ser ArgMet Cys Val Gly Lys Glu Phe Ala Lys Val Leu Leu 435 440 445 Lys Ile PheLeu Val Glu Leu Thr Gln His Cys Asn Trp Ile Leu Ser 450 455 460 Asn GlyPro Pro Thr Met Lys Thr Gly Pro Thr Ile Tyr Pro Val Asp 465 470 475 480Asn Leu Pro Thr Lys Phe Thr Ser Tyr Val Arg Asn 485 490 24 504 PRT Homosapiens 24 Met Ala Leu Ile Pro Asp Leu Ala Met Glu Thr Trp Leu Leu LeuAla 1 5 10 15 Val Ser Leu Val Leu Leu Tyr Leu Tyr Gly Thr His Ser HisGly Leu 20 25 30 Phe Lys Lys Leu Gly Ile Pro Gly Pro Thr Pro Leu Pro PheLeu Gly 35 40 45 Asn Ile Leu Ser Tyr His Lys Gly Phe Cys Met Phe Asp MetGlu Cys 50 55 60 His Lys Lys Tyr Gly Lys Val Trp Gly Phe Tyr Asp Gly GlnGln Pro 65 70 75 80 Val Leu Ala Ile Thr Asp Pro Asp Met Ile Lys Leu ValLeu Val Lys 85 90 95 Glu Cys Tyr Ser Val Phe Thr Asn Arg Glu Pro Phe GlyPro Val Gly 100 105 110 Phe Met Lys Ser Ala Ile Ser Ile Ala Glu Asp GluGlu Trp Lys Arg 115 120 125 Leu Arg Ser Leu Leu Ser Pro Thr Phe Thr SerGly Lys Leu Lys Glu 130 135 140 Met Val Pro Ile Ile Ala Gln Tyr Gly AspVal Leu Val Arg Asn Leu 145 150 155 160 Arg Arg Glu Arg Glu Thr Gly LysPro Val Thr Leu Lys Asp Val Phe 165 170 175 Gly Ala Tyr Ser Met Asp ValIle Thr Ser Ser Ser Phe Gly Val Asn 180 185 190 Val Asp Ser Leu Asn AsnPro Gln Asp Pro Leu Val Glu Asn Thr Lys 195 200 205 Lys Leu Leu Arg PheAsp Phe Leu Asp Pro Phe Phe Leu Ser Ile Thr 210 215 220 Val Phe Pro PheLeu Ile Pro Ile Leu Glu Val Leu Asn Ile Cys Val 225 230 235 240 Phe ProArg Glu Val Thr Asn Phe Leu Arg Lys Ala Val Lys Arg Met 245 250 255 LysGlu Ser Arg Leu Glu Asp Thr Gln Lys His Arg Val Asp Phe Leu 260 265 270Gln Leu Met Ile Asp Ser His Lys Asn Ser Lys Glu Thr Glu Ser His 275 280285 Lys Ala Leu Ser Asp Leu Glu Leu Val Ala Gln Ser Ile Ile Phe Ile 290295 300 Phe Ala Gly Tyr Glu Thr Thr Ser Ser Val Leu Ser Phe Ile Met Tyr305 310 315 320 Glu Leu Ala Thr His Pro Asp Val Gln Gln Lys Leu Gln GluGlu Ile 325 330 335 Asp Ala Val Leu Pro Asn Lys Ala Pro Pro Thr Tyr AspThr Val Leu 340 345 350 Gln Met Glu Tyr Leu Asp Met Val Val Asn Glu ThrLeu Arg Leu Phe 355 360 365 Pro Ile Ala Met Arg Leu Glu Arg Val Cys LysLys Asp Val Glu Ile 370 375 380 Asn Gly Met Phe Ile Pro Lys Gly Trp ValVal Met Ile Pro Ser Tyr 385 390 395 400 Ala Leu His Arg Asp Pro Lys TyrTrp Thr Glu Pro Glu Lys Phe Leu 405 410 415 Pro Glu Arg Phe Ser Lys LysAsn Lys Asp Asn Ile Asp Pro Tyr Ile 420 425 430 Tyr Thr Pro Phe Gly SerGly Pro Arg Asn Cys Ile Gly Met Arg Phe 435 440 445 Ala Leu Met Asn MetLys Leu Ala Leu Ile Arg Val Leu Gln Asn Phe 450 455 460 Ser Phe Lys ProCys Lys Glu Thr Gln Ile Pro Leu Lys Leu Ser Leu 465 470 475 480 Gly GlyLeu Leu Gln Pro Glu Lys Pro Val Val Leu Lys Val Glu Ser 485 490 495 ArgAsp Gly Thr Val Ser Gly Ala 500 25 575 PRT Artificial Sequence Consensussequence 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Leu Leu Ser Xaa Xaa Ala Leu XaaVal Xaa 35 40 45 Leu Xaa Leu Ala Ala Arg Arg Xaa Xaa Xaa Arg Tyr Xaa XaaXaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Arg Arg Lys Xaa Leu Pro Pro Gly Thr MetGly Leu 65 70 75 80 Pro Xaa Leu Gly Glu Thr Leu Gln Phe Leu Lys Xaa XaaXaa Xaa Xaa 85 90 95 Xaa Pro Gly Asp Phe Xaa Lys Glu Arg Val Xaa Xaa TyrGly Xaa Xaa 100 105 110 Xaa Xaa Ile Tyr Lys His Leu Phe Gly Glu Pro ThrIle Xaa Ser Xaa 115 120 125 Asp Ala Glu Leu Asn Arg Phe Xaa Leu Xaa AsnGlu Gly Xaa Lys Leu 130 135 140 Phe Xaa Cys Xaa Xaa Pro Ala Ser Xaa XaaGly Xaa Leu Gly Lys Xaa 145 150 155 160 Ser Leu Xaa Ala Xaa Xaa Gly XaaGlu His Lys Arg Met Arg Xaa Leu 165 170 175 Leu Xaa Ser Xaa Phe Ser XaaXaa Xaa Xaa Leu Asp His Xaa Leu Pro 180 185 190 Xaa Ile Asp Arg Xaa ValArg Ser Xaa Leu Xaa Xaa Trp Xaa Xaa Xaa 195 200 205 Xaa Gln Lys Xaa XaaIle Val Xaa Xaa Xaa Xaa Glu Xaa Lys Lys Met 210 215 220 Thr Phe Asp XaaXaa Xaa Lys Xaa Xaa Met Gly Xaa Xaa Pro Xaa Xaa 225 230 235 240 Glu XaaThr Xaa Xaa Xaa Xaa Leu Val Xaa Glu Xaa Glu Xaa Leu Ile 245 250 255 LysGly Leu Phe Ser Leu Pro Ile Asn Leu Pro Xaa Thr Ala Tyr Xaa 260 265 270Lys Ala Leu Xaa Ala Arg Ala Phe Xaa Xaa Ala Xaa Leu Glu Xaa Xaa 275 280285 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ile Xaa Glu Xaa Arg Xaa Glu Glu 290295 300 Glu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa305 310 315 320 Xaa Xaa Xaa Xaa Xaa Xaa Asp Asp Leu Leu Gly Leu Leu XaaAla Xaa 325 330 335 Xaa Xaa Xaa Xaa Xaa Glu Asp Glu Xaa Xaa Xaa Xaa LeuSer Asp Xaa 340 345 350 Glu Ile Xaa Asp Xaa Ile Xaa Xaa Leu Leu Phe AlaGly His Glu Thr 355 360 365 Thr Ser Ser Xaa Leu Xaa Xaa Ala Val Lys PheLeu Xaa Glu His Pro 370 375 380 Asp Val Xaa Glu Xaa Leu Arg Glu Glu HisXaa Ala Ile Xaa Arg Ala 385 390 395 400 Lys Lys Xaa Xaa Xaa Glu Ser XaaLeu Thr Xaa Xaa Asp Tyr Lys Lys 405 410 415 Met Xaa Tyr Thr Xaa Cys ValIle Asn Glu Thr Leu Arg Leu Ala Xaa 420 425 430 Ile Val Gly Gly Xaa PheArg Xaa Ala Xaa Lys Asp Val Glu Ile Asn 435 440 445 Gly Tyr Xaa Ile ProLys Gly Trp Lys Val Xaa Tyr Ser Ile Arg Ala 450 455 460 Val His Leu AspPro Asp Xaa Tyr Pro Asp Pro Glu Lys Phe Asn Pro 465 470 475 480 Xaa ArgTrp Xaa Xaa Lys Xaa Xaa Xaa Xaa Ser Asn Ser Xaa Xaa Xaa 485 490 495 XaaXaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Pro Phe Gly Gly Gly Pro 500 505 510Arg Leu Cys Pro Gly Lys Glu Leu Ala Lys Leu Glu Met Xaa Val Phe 515 520525 Leu His Arg Leu Val Gln Xaa Phe Trp Glu Leu Ala Xaa Xaa Xaa Asp 530535 540 Xaa Xaa Xaa Lys Leu Val Xaa Phe Pro Thr Xaa Arg Pro Xaa Asp Asn545 550 555 560 Leu Pro Ile Lys Val Xaa Xaa Arg Asp Xaa Xaa Xaa Xaa XaaXaa 565 570 575 26 11 PRT Artificial Sequence Heme binding domain 26 ProPhe Gly Xaa Gly Arg Arg Xaa Cys Xaa Gly 1 5 10 27 14 PRT ArtificialSequence Heme binding domain 27 Pro Phe Gly Gly Phe Pro Arg Leu Cys ProGly Lys Glu Leu 1 5 10 28 17 PRT Artificial Sequence Signature sequence28 Xaa Leu Leu Phe Ala Gly His Glu Thr Thr Ser Ser Xaa Ile Xaa Xaa 1 510 15 Ala 29 11 PRT Artificial Sequence Exemplary sequence 29 Pro PheGly Gly Gly Pro Arg Leu Cys Ala Gly 1 5 10 30 6 PRT Arabidopsis thaliana30 Ala Gly His Glu Thr Ser 1 5

1.-16. (Cancelled).
 17. A method of modulating a DWF4 polypeptidecomprising: (a) providing a host cell, wherein said host cell comprisesa recombinant vector, said recombinant vector comprising: (i) anisolated dwf 4 polynucleotide, wherein said isolated dwf4 polynucleotidecomprises a sequence having at least 50% identity to SEQ ID NO:1, andcomplements and reverse complements thereof; and (ii) a control elementoperably linked to said isolated dwf4 polynucleotide, whereby a codingsequence within said isolated dwf4 polynucleotide can be transcribed andtranslated in said host cell; and (b) culturing said host cell underconditions whereby said isolated dwf4 polynucleotide is transcribed,wherein expression of dwf4 is inhibited. 18.-35. (Cancelled).
 36. Amethod for producing a transgenic plant having an altered phenotyperelative to a corresponding wild-type plant comprising: introducing anisolated dwf 4 polynucleotide into a plant cell, wherein said isolateddwf4 polynucleotide comprises a sequence having at least 50% identity toSEQ ID NO:1, and complements and reverse complements thereof; andproducing a transgenic plant from said plant cell, said transgenic planthaving an altered phenotype relative to the wild-type plant, wherein theisolated dwf4 polynucleotide inhibits expression of dwf4.
 37. A methodfor producing a transgenic plant having an altered phenotype relative toa corresponding wild-type plant comprising: introducing first and secondisolated dwf 4 polynucleotides into a plant cell, wherein said first andsecond isolated dwf4 polynucleotides independently comprise a sequencehaving at least 50% identity to SEQ ID NO:1. and complements and reversecomplements thereof; said first and second isolated dwf4 polynucleotidesoperably linked to at least first and second tissue-specific promoters,wherein said first isolated dwf4 polynucleotide is overexpressed andwherein said second isolated dwf4 polynucleotide inhibits expression ofdwf4; and producing a transgenic plant from said plant cell, saidtransgenic plant having an altered phenotype relative to the wild-typeplant. 38.-57. (Cancelled).